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A novel point mutation in RpoB improves osmotolerance and succinic acid production in Escherichia coli.

ABSTRACT: BACKGROUND:Escherichia coli suffer from osmotic stress during succinic acid (SA) production, which reduces the performance of this microbial factory. RESULTS:Here, we report that a point mutation leading to a single amino acid change (D654Y) within the ?-subunit of DNA-dependent RNA polymerase (RpoB) significantly improved the osmotolerance of E. coli. Importation of the D654Y mutation of RpoB into the parental strain, Suc-T110, increased cell growth and SA production by more than 40% compared to that of the control under high glucose osmolality. The transcriptome profile, determined by RNA-sequencing, showed two distinct stress responses elicited by the mutated RpoB that counterbalanced the osmotic stress. Under non-stressed conditions, genes involved in the synthesis and transport of compatible solutes such as glycine-betaine, glutamate or proline were upregulated even without osmotic stimulation, suggesting a "pre-defense" mechanism maybe formed in the rpoB mutant. Under osmotic stressed conditions, genes encoding diverse sugar transporters, which should be down-regulated in the presence of high osmotic pressure, were derepressed in the rpoB mutant. Additional genetic experiments showed that enhancing the expression of the mal regulon, especially for genes that encode the glycoporin LamB and maltose transporter, contributed to the osmotolerance phenotype. CONCLUSIONS:The D654Y single amino acid substitution in RpoB rendered E. coli cells resistant to osmotic stress, probably due to improved cell growth and viability via enhanced sugar uptake under stressed conditions, and activated a potential "pre-defense" mechanism under non-stressed conditions. The findings of this work will be useful for bacterial host improvement to enhance its resistance to osmotic stress and facilitate bio-based organic acids production.

SUBMITTER: Xiao M

PROVIDER: S-EPMC5307762 | biostudies-literature | 2017 Feb

REPOSITORIES: biostudies-literature

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A novel point mutation in RpoB improves osmotolerance and succinic acid production in Escherichia coli.

Xiao Mengyong M Zhu Xinna X Xu Hongtao H Tang Jinlei J Liu Ru R Bi Changhao C Fan Feiyu F Zhang Xueli X

BMC biotechnology 20170213 1

<h4>Background</h4>Escherichia coli suffer from osmotic stress during succinic acid (SA) production, which reduces the performance of this microbial factory.<h4>Results</h4>Here, we report that a point mutation leading to a single amino acid change (D654Y) within the β-subunit of DNA-dependent RNA polymerase (RpoB) significantly improved the osmotolerance of E. coli. Importation of the D654Y mutation of RpoB into the parental strain, Suc-T110, increased cell growth and SA production by more than ...[more]

PMID: 28193207

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Project description:Purpose: To understand how D654Y mutated RpoB affect gene expression under either normal or osmotic stress conditions in Escherichia coli . Methods: RNA-seq libraries were constructed for four samples, including (I) The parental strain Suc-T110 grown at normal condition (5%w/v glucose); (II)RpoB mutant RpoBD645Y [Suc-T110::rpoB (D654Y), RNA-seq library named NZ-502] grown at normal condition;(III) Suc-T110 grown at osmotic stress condition(12%w/v glucose);(IV) NZ-502 grown at osmotic stress condition. For preparation of RNA samples, Fresh colonies were picked from New NBS mineral salts plates containing 20 g liter-1 glucose, inoculated into 250 ml flasks containing 30 ml NBS mineral salts medium with 20 g liter-1 glucose, and grown at 37°C and 250 rpm for 12 h. Cultures were then transferred to a 500 ml fermentation vessel containing 250 ml NBS mineral salts medium with either 5%w/v or 12%w/v glucose. After 48 h, 1 ml culture was harvested by centrifugation, frozen in liquid nitrogen and sent to Beijing Genomics Institute (BGI, Shenzhen, China). The four 90-nt paired-end RNA-seq libraries were generated commercially at Beijing Genomics Institute by using the HiSeq™ 2000 platform. Sequencing data was handled essentially with Bowtie2, NOIseq. Expression levels are presented as Reads Per Kilobase of transcript per Million mapped reads (RPKM). Results: we first comparative accessed expression levels of Suc-T110 and NZ-502 under normal condition. Among the 4200 predicted genes, 128 genes were found altered their expression in NZ502 compared with Suc-T110; with 38 targets specific up-regulated while 90 targets down-regulated. Genes engaged in biological function of amino acid metabolism (P-value=4.60E-05) were significantly enriched among in induced gene set. Under osmotic stress condition, 244 differential expressed genes were identified in NZ-502 when compared with Suc-T110, with 132 targets specific up-regulated while 112 targets down-regulated, Functional enrichment of 132 up regulated genes showed the most significantly enriched physiological function of NZ-502 upon hyperosmotic stress was associated with carbon source transportation (FunCat term: 20.01.03 C-compound and carbohydrate transport, P-value=9.51E-05), which containing a group of genes encoding diverse sugar transporters. Conclusions: Transcriptome profiling suggested that derepression of sugar transporters might be the primary cause for NZ-502 to resist osmotic stress.

Project description:BACKGROUND: Hydroxycinnamates (HCs) are mainly produced in plants. Caffeic acid (CA), p-coumaric acid (PA), ferulic acid (FA) and sinapic acid (SA) are members of the HC family. The consumption of HC by human might prevent cardiovascular disease and some types of cancer. The solubility of HCs is increased through thioester conjugation to various compounds such as quinic acid, shikimic acid, malic acid, anthranilic acid, and glycerol. Although hydroxycinnamate conjugates can be obtained from diverse plant sources such as coffee, tomato, potato, apple, and sweet potato, some parts of the world have limited availability to these compounds. Thus, there is growing interest in producing HC conjugates as nutraceutical supplements. RESULTS: Hydroxycinnamoyl transferases (HCTs) including hydroxycinnamate-CoA shikimate transferase (HST) and hydroxycinnamate-CoA quinate transferase (HQT) were co-expressed with 4-coumarateCoA:ligase (4CL) in Escherichia coli cultured in media supplemented with HCs. Two hydroxycinnamoyl conjugates, p-coumaroyl shikimates and chlorogenic acid, were thereby synthesized. Total 29.1 mg/L of four different p-coumaroyl shikimates (3-p-coumaroyl shikimate, 4-p-coumaroyl shikimate, 3,4-di-p-coumaroyl shikimate, 3,5-di-p-coumaroyl shikimate, and 4,5-di-p-coumaroyl shikimate) was obtained and 16 mg/L of chlorogenic acid was synthesized in the wild type E. coli strain. To increase the concentration of endogenous acceptor substrates such as shikimate and quinate, the shikimate pathway in E. coli was engineered. A E. coli aroL and aroK gene were mutated and the resulting mutants were used for the production of p-coumaroyl shikimate. An E. coli aroD mutant was used for the production of chlorogenic acid. We also optimized the vector and cell concentration optimization. CONCLUSIONS: To produce p-coumaroyl-shikimates and chlorogenic acid in E. coli, several E. coli mutants (an aroD mutant for chlorogenic acid production; an aroL, aroK, and aroKL mutant for p-coumaroyl-shikimates production) were made and each mutant was tested using an optimized construct. Using this strategy, we produced 235 mg/L of p-coumaroyl-shikimates and 450 mg/L of chlorogenic acid.

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A novel point mutation in RpoB improves osmotolerance and succinic acid production in Escherichia coli.

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A novel point mutation in RpoB improves osmotolerance and succinic acid production in Escherichia coli.

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