A high‐throughput screen for hyaluronic acid accumulation in recombinant Escherichia coli transformed by libraries of engineered sigma factors

Abstract

Hyaluronic acid (HA) is an important biomaterial with functional medical and cosmetic applications. As its synthesis has been recently reported in recombinant bacteria, it is of interest to develop a high throughput screening method for the rapid isolation of HA accumulating strains transformed by combinatorial libraries. Here we report a novel two-step screening strategy to select for better HA-producing recombinant Escherichia coli strains transformed by mutation libraries of rpoD and rpoS, coding for the sigma(D) and sigma(S) factors of the RNA polymerase, respectively. The first screen, based on translucent colony morphology identification, was used to qualitatively distinguish HA-producing strains on agar plates from non-HA producing strains that exhibit dense colony morphology. The second screen was based on the photometric measurement of an alcian blue staining solution that precipitates with HA, creating an inverse relationship between HA concentration and alcian blue absorbance. The color attenuation fitted a second-order polynomial between HA concentration and OD(540) absorbance. Using the alcian blue absorbance quantification, 74 translucent colonies from the HA-rpoD library and 78 translucent colonies from the HA-rpoS library were isolated and cultured for optimal strain selection. Three representative superior recombinants with high, medium and low increase of HA accumulation, respectively, were identified by the screen from the HA-rpoD and HA-rpoS mutant library. Further flask culture confirmed that results of the library screen were reliable and the superior recombinant D72 highly accumulated HA of 561.4 mg/L with a productivity of approximately 265 mg HA/g dry cell. Sequencing results showed that the mutant rpoD gene in D72 is in a truncated protein that lacks the conserved regions 3 and 4 of the sigma(D). Generally, this two-step high throughput screen presents a promising strategy for selecting superior HA-producing strains from large scale mutation libraries.

Mutagenesis of the Bacterial RNA Polymerase Alpha Subunit for Improvement of Complex Phenotypes

“…During our studies of HA production, rpoA mutants showed up to ϳ60% improvement in titer (Fig. 4) compared to ϳ40% in the case of rpoD as measured by the same method (48). Despite these results, we do not believe that this constitutes sufficient evidence for favoring one target or the other; instead, we have argued that a better measure of the usefulness of a library is related to the a priori probability of finding improved mutants (22), not to whether a particular target delivers a phenotype of interest or not.…”

“…Using a previously reported high-throughput screening platform (48), we were able to screen an rpoA library and isolate several improved rpoA mutants. Figure 5 shows the HA concentrations relative to the control, quantified using the alcian blue method as described previously (48). It is clear from these results that significant phenotypic diversity was generated through the introduction of the rpoA library.…”

“…Mutant libraries of HA-producing strain E. coli Top10/ (pMBAD-sseABC, pHACm-rpoA) transformed with the rpoA libraries were first screened by a translucent colony phenotype and then selected by alcian blue staining as described previously (48). E. coli Top10/(pMBAD-sseABC) was used as a control and, in total, 76 translucent colonies in the library were stained by alcian blue.…”

“…Random approaches for strain engineering have been extensively employed for the improvement of complex or poorly understood phenotypes, such as metabolite overproduction or tolerance to toxic compounds (33,34,39,48). Chemical or physical mutagenesis of the genome followed by screening has been a traditional means of improving several phenotypes, particularly for increasing antibiotic production in the pharmaceutical industry (10,11).…”

Reverse biological engineering of hrdB to enhance the production of avermectins in an industrial strain of Streptomyces avermitilis

“…It is suspected that a cascade of other sigma factors, pleiotropic regulators and pathway-specific regulators, might be triggered to express by σ hrdB (32). Engineering the binding factors of RNA polymerase has been successfully applied in the improvement of ethanol tolerance and productivity in Saccharomyces cerevisiae (33), and in the yield improvement of products expressed in the heterogeneous host E. coli (34,35). By binding to and affecting the promoter specificity of the RNA polymerase core enzyme, σ hrdB directs the selective transcription of gene sets and acts cooperatively with one or more Streptomyces extracytoplasmic function sigma factors to exert its control on downstream targets.…”