Efficient synthesis of L-lactic acid from glycerol by metabolically engineered Escherichia coli

Due to its abundance and low-price, glycerol has become an attractive carbon source for the industrial production of value-added fuels and chemicals. This work reports the engineering of E. coli for the efficient conversion of glycerol into L-lactic acid(L-lactate). Escherichia coli strains have previously been metabolically engineered for the microaerobic production of D-lactic acid from glycerol in defined media by disrupting genes that minimize the synthesis of succinate, acetate, and ethanol, and also overexpressing the respiratory route of glycerol dissimilation (GlpK/GlpD). Here, further rounds of rationale design were performed on these strains for the homofermentative production of L-lactate, not normally produced in E. coli. Specifically, L-lactate production was enabled by: 1), replacing the native D-lactate specific dehydrogenase with Streptococcus bovis L-lactate dehydrogenase (L-LDH), 2) blocking the methylglyoxal bypass pathways to avoid the synthesis of a racemic mixture of D- and L-lactate and prevent the accumulation of toxic intermediate, methylglyoxal, and 3) the native aerobic L-lactate dehydrogenase was blocked to prevent the undesired utilization of L-lactate. The engineered strain produced 50 g/L of L-lactate from 56 g/L of crude glycerol at a yield 93% of the theoretical maximum and with high optical (99.9%) and chemical (97%) purity. This study demonstrates the efficient conversion of glycerol to L-lactate, a microbial process that had not been reported in the literature prior to our work. The engineered biocatalysts produced L-lactate from crude glycerol in defined minimal salts medium at high chemical and optical purity

Transcription from bacteriophage λ pR promoter is regulated independently and antagonistically by DksA and ppGpp

The stringent response effector, guanosine tetraphosphate (ppGpp), adjust gene expression and physiology in bacteria, by affecting the activity of various promoters. RNA polymerase-interacting protein, DksA, was proposed to be the co-factor of ppGpp effects; however, there are reports suggesting independent roles of these regulators. Bacteriophage λ major lytic promoter, pR, is down-regulated by the stringent response and ppGpp. Here, we present evidence that DksA significantly stimulates pR-initiated transcription in vitro in the reconstituted system. DksA is also indispensable for pR activity in vivo. DksA-mediated activation of pR-initiated transcription is predominant over ppGpp effects in the presence of both regulators in vitro. The possible role of the opposite regulation by ppGpp and DksA in λ phage development is discussed. The major mechanism of DksA-mediated activation of transcription from pR involves facilitating of RNA polymerase binding to the promoter region, which results in more productive transcription initiation. Thus, our results provide evidence for the first promoter inhibited by ppGpp that can be stimulated by the DksA protein both in vivo and in vitro. Therefore, DksA role could be not only independent but antagonistic to ppGpp in transcription regulation

Transcriptional Infidelity Promotes Heritable Phenotypic Change in a Bistable Gene Network

Bistable epigenetic switches are fundamental for cell fate determination in unicellular and multicellular organisms. Regulatory proteins associated with bistable switches are often present in low numbers and subject to molecular noise. It is becoming clear that noise in gene expression can influence cell fate. Although the origins and consequences of noise have been studied, the stochastic and transient nature of RNA errors during transcription has not been considered in the origin or modeling of noise nor has the capacity for such transient errors in information transfer to generate heritable phenotypic change been discussed. We used a classic bistable memory module to monitor and capture transient RNA errors: the lac operon of Escherichia coli comprises an autocatalytic positive feedback loop producing a heritable all-or-none epigenetic switch that is sensitive to molecular noise. Using single-cell analysis, we show that the frequency of epigenetic switching from one expression state to the other is increased when the fidelity of RNA transcription is decreased due to error-prone RNA polymerases or to the absence of auxiliary RNA fidelity factors GreA and GreB (functional analogues of eukaryotic TFIIS). Therefore, transcription infidelity contributes to molecular noise and can effect heritable phenotypic change in genetically identical cells in the same environment. Whereas DNA errors allow genetic space to be explored, RNA errors may allow epigenetic or expression space to be sampled. Thus, RNA infidelity should also be considered in the heritable origin of altered or aberrant cell behaviour

Transcription initiation factor DksA has diverse effects on RNA chain elongation

Bacterial transcription factors DksA and GreB belong to a family of coiled-coil proteins that bind within the secondarychannel of RNA polymerase (RNAP). These proteins display structural homology but play different regulatory roles. DksA disrupts RNAP interactions with promoter DNA and inhibits formation of initiation complexes, sensitizing rRNA synthesis to changes in concentrations of ppGpp and NTPs. Gre proteins remodel the RNAP active site and facilitate cleavage of the nascent RNA in elongation complexes. However, DksA and GreB were shown to have overlapping effects during initiation, and in vivo studies suggested that DksA may also function at post-initiation steps. Here we show that DksA has many features of an elongation factor: it inhibits both RNA chain extension and RNA shortening by exonucleolytic cleavage or pyrophosphorolysis and increases intrinsic termination in vitro and in vivo. However, DksA has no effect on Rho- or Mfd-mediated RNA release or nascent RNA cleavage in backtracked complexes, the regulatory target of Gre factors. Our results reveal that DksA effects on elongating RNAP are very different from those of GreB, suggesting that these regulators recognize distinct states of the transcription complex

A hyper-mutant of the unusual σ70-Pr promoter bypasses synergistic ppGpp/DksA co-stimulation

The activities of promoters can be temporally and conditionally regulated by mechanisms other than classical DNA-binding repressors and activators. One example is the inherently weak σ70-dependent Pr promoter that ultimately controls catabolism of phenolic compounds. The activity of Pr is up-regulated through the joint action of ppGpp and DksA that enhance the performance of RNA polymerase at this promoter. Here, we report a mutagenesis analysis that revealed substantial differences between Pr and other ppGpp/DksA co-stimulated promoters. In vitro transcription and RNA polymerase binding assays show that it is the T at the −11 position of the extremely suboptimal −10 element of Pr that underlies both poor binding of σ70-RNAP and a slow rate of open complex formation—the process that is accelerated by ppGpp and DksA. Our findings support the idea that collaborative action of ppGpp and DksA lowers the rate-limiting transition energy required for conversion between intermediates on the road to open complex formation

Recombination Phenotypes of Escherichia coli greA Mutants

<p>Abstract</p> <p>Background</p> <p>The elongation factor GreA binds to RNA polymerase and modulates transcriptional pausing. Some recent research suggests that the primary role of GreA may not be to regulate gene expression, but rather, to promote the progression of replication forks which collide with RNA polymerase, and which might otherwise collapse. Replication fork collapse is known to generate dsDNA breaks, which can be recombinogenic. It follows that GreA malfunction could have consequences affecting homologous recombination.</p> <p>Results</p> <p><it>Escherichia coli </it>mutants bearing substitutions of the active site acidic residues of the transcription elongation factor GreA, D41N and E44K, were isolated as suppressors of growth inhibition by a toxic variant of the bacteriophage lambda Red-beta recombination protein. These mutants, as well as a D41A <it>greA </it>mutant and a <it>greA </it>deletion, were tested for proficiency in recombination events. The mutations were found to increase the efficiency of RecA-RecBCD-mediated and RecA-Red-mediated recombination, which are replication-independent, and to decrease the efficiency of replication-dependent Red-mediated recombination.</p> <p>Conclusion</p> <p>These observations provide new evidence for a role of GreA in resolving conflicts between replication and transcription.</p

TraR, a Homolog of a RNAP Secondary Channel Interactor, Modulates Transcription

Recent structural and biochemical studies have identified a novel control mechanism of gene expression mediated through the secondary channel of RNA Polymerase (RNAP) during transcription initiation. Specifically, the small nucleotide ppGpp, along with DksA, a RNAP secondary channel interacting factor, modifies the kinetics of transcription initiation, resulting in, among other events, down-regulation of ribosomal RNA synthesis and up-regulation of several amino acid biosynthetic and transport genes during nutritional stress. Until now, this mode of regulation of RNAP was primarily associated with ppGpp. Here, we identify TraR, a DksA homolog that mimics ppGpp/DksA effects on RNAP. First, expression of TraR compensates for dksA transcriptional repression and activation activities in vivo. Second, mutagenesis of a conserved amino acid of TraR known to be critical for DksA function abolishes its activity, implying both structural and functional similarity to DksA. Third, unlike DksA, TraR does not require ppGpp for repression of the rrnB P1 promoter in vivo and in vitro or activation of amino acid biosynthesis/transport genes in vivo. Implications for DksA/ppGpp mechanism and roles of TraR in horizontal gene transfer and virulence are discussed

A Synthetic Biology Approach to Engineer a Functional Reversal of the β‑Oxidation Cycle

While we have recently constructed a functional reversal of the β-oxidation cycle as a platform for the production of fuels and chemicals by engineering global regulators and eliminating native fermentative pathways, the system-level approach used makes it difficult to determine which of the many deregulated enzymes are responsible for product synthesis. This, in turn, limits efforts to fine-tune the synthesis of specific products and prevents the transfer of the engineered pathway to other organisms. In the work reported here, we overcome the aforementioned limitations by using a synthetic biology approach to construct and functionally characterize a reversal of the β-oxidation cycle. This was achieved through the <i>in vitro</i> kinetic characterization of each functional unit of the core and termination pathways, followed by their <i>in vivo</i> assembly and functional characterization. With this approach, the four functional units of the core pathway, thiolase, 3-hydroxyacyl-CoA dehydrogenase, enoyl-CoA hydratase/3-hydroxyacyl-CoA dehydratase, and acyl-CoA dehydrogenase/trans-enoyl-CoA reductase, were purified and kinetically characterized <i>in vitro</i>. When these four functional units were assembled <i>in vivo</i> in combination with thioesterases as the termination pathway, the synthesis of a variety of 4-C carboxylic acids from a one-turn functional reversal of the β-oxidation cycle was realized. The individual expression and modular construction of these well-defined core components exerted the majority of control over product formation, with only highly selective termination pathways resulting in shifts in product formation. Further control over product synthesis was demonstrated by overexpressing a long-chain thiolase that enables the operation of multiple turns of the reversal of the β-oxidation cycle and hence the synthesis of longer-chain carboxylic acids. The well-defined and self-contained nature of each functional unit makes the engineered reversal of the β-oxidation cycle “chassis neutral” and hence transferrable to the host of choice for efficient fuel or chemical production