Physiological characterization and quantitative proteomic analyses of metabolically engineered E. coli K4 strains with improved pathways for capsular polysaccharide biosynthesis

Abstract

Among capsulated bacteria, some produce polysaccharides with unique properties that have been shown to possess relevant industrial applications and commercial value. The capsular polysaccharide (CPS) produced by Escherichia coli K4 is similar to chondroitin sulphate, and recent efforts focused on the development of genetic and fermentation strategies to increase its production titers up to technologically attractive levels. However, the control of the metabolic pathways leading to CPS synthesis together with the effect of varying the concentration of pathway intermediates on CPS final titers, is still quite unexplored, and not fully understood. In the present study four genes involved in the biosynthesis of UDP-sugar CPS precursors, namely kfoA, kfoF, pgm, and galU, were overexpressed in different combinations, and diversely affected the biosynthetic machinery. At the physiological level, results revealed a central role for kfoF, coding for UDP-glucose dehydrogenase, that increased CPS production mostly. In the attempt to unravel the molecular mechanisms regulating CPS biosynthesis, an in depth analysis of the proteome of the recombinant strains overexpressing respectively pgm and galU, and pgm, galU, and kfoF was performed and compared to the wild-type. Although, interestingly, in both strains the impact of the genetic manipulation seemed rather limited at the proteome level, results obtained from the triple mutant indicated a crosstalk between the two pathways leading to UDP-sugar precursors biosynthesis, and also an unexpected link with the purine biosynthetic pathway. Overall our results present new insights into the role of metabolic intermediates for the formation of capsular polysaccharides, utilizing a systematic approach of metabolic engineering, combined with state-of-the-art quantitative proteomic approaches, as well as genetic and physiological information.

Production and purification of higher molecular weight chondroitin by metabolically engineered Escherichia coli K4 strains

“…This protocol was then used for the analysis of E.coli K4, a natural producer of CS-like polysaccharide, and four recombinant derivates thereof, overexpressing endogenous genes involved in the biosynthesis of UDP-sugar CPS precursors ( Table 1). The construction and a very basic characterization of all these strains with respect to CPS production levels were already previously described 12 . In this study we extended the physiological characterization of these strains in well-controlled bioreactors also to larger scales (up to 22 L), as it has been already reported that different cultivation parameters like fermentation duration, but also in particular scale dependent factors like mixing can impact the molecular weight of the produced biopolymers 13,14 .…”

“…The wild type strain used in all experiments was E.coli K4 serotype O5:K4:H4 (CCUG 11307), purchased from the Culture Collection University of Goteborg. The genetic characteristics and intended metabolic targets of recombinant strains 12 used in the work are reported in Table 1 . For all recombinant strains kanamycin was supplemented in the solid (50 µg/mL) and liquid media used for all experiments.…”

“…We previously characterized in shake flasks different recombinant strains that overexpress genes involved in the last steps of UDP-GalNAc and UDP-GlcA biosynthesis, namely kfoA , kfoF , pgm , and galU 12 . All strains demonstrated increased CPS titers, however major improvements were encountered in those overexpressing the UDP-glucose dehydrogenase, encoded by kfoF , either alone or in combination 12 . Previous studies in other hosts also demonstrated that UDP-glucose dehydrogenase is a rate limiting enzyme in the production of GAG and GAG-like polysaccharides 13 – 17 and in recombinant Bacillus subtilis its overexpression increased the Mw of the produced polymer by 35%; however, in this case the polymer size was almost reduced by half when scaling the process from shake flasks to 3 L bioreactors, due, according to the authors, to the shear stress caused by mechanical agitation 13 .…”

“…The aim of the present work was, therefore, to investigate whether the previously described 12 metabolic engineering strategies affected the molecular weight of the produced polymers, and to potentially correlate this to the modification of intracellular metabolite pools in order to elucidate the regulatory network of K4 CPS quantity and quality on multiple cellular levels. Fed-batch processes up to the 22 L scale were used to evaluate the performance of the different strains and to determine polymer size in potentially industrially applicable controlled fermentation conditions.…”

“…We previously characterized in shake flasks different recombinant strains that overexpress genes involved in the last steps of UDP-GalNAc and UDP-GlcA biosynthesis, namely kfoA, kfoF, pgm, and galU 12 . All strains demonstrated increased CPS titers, however major improvements were encountered in those overexpressing the UDP-glucose dehydrogenase, encoded by kfoF, either alone or in combination 12 .…”