Alternative carbohydrate pathways - enzymes, functions and engineering.

Metabolic engineering is crucial in the development of production strains for platform chemicals, pharmaceuticals and biomaterials from renewable resources. The central carbon metabolism (CCM) of heterotrophs plays an essential role in the conversion of biomass to the cellular building blocks required for growth. Yet, engineering the CCM ultimately aims toward a maximization of flux toward products of interest. The most abundant dissimilative carbohydrate pathways amongst prokaryotes (and eukaryotes) are the Embden-Meyerhof-Parnas (EMP) and the Entner-Doudoroff (ED) pathways, which build the basics for heterotrophic metabolic chassis strains. Although the EMP is regarded as the textbook example of a carbohydrate pathway owing to its central role in production strains like Escherichia coli, Saccharomyces cerevisiae and Bacillus subtilis, it is either modified, complemented or even replaced by alternative carbohydrate pathways in different organisms. The ED pathway also plays key roles in biotechnological relevant bacteria, like Zymomonas mobilis and Pseudomonas putida, and its importance was recently discovered in photoautotrophs and marine microorganisms. In contrast to the EMP, the ED pathway and its variations are not evolutionary optimized for high ATP production and it differs in key principles such as protein cost, energetics and thermodynamics, which can be exploited in the construction of unique metabolic designs. Single ED pathway enzymes and complete ED pathway modules have been used to rewire carbon metabolisms in production strains and for the construction of cell-free enzymatic pathways. This review focuses on the differences of the ED and EMP pathways including their variations and discusses the use of alternative pathway strategies for in vivo and cell-free metabolic engineering.

The role of the Synechocystis sp. PCC 6803 homolog of the circadian clock output regulator RpaA in day-night transitions.

Cyanobacteria exhibit rhythmic gene expression with a period length of 24 hours to adapt to daily environmental changes. In the model organism Synechococcuselongatus PCC 7942, the central oscillator consists of the three proteins KaiA, KaiB and KaiC and utilizes the histidine kinase SasA and its response regulator RpaA as output-signaling pathway. Synechocystis sp. PCC 6803 contains in addition to the canonical kaiAB1C1 gene cluster two further homologs of the kaiB and kaiC genes. Here, we demonstrate that the SasA-RpaA system interacts with the KaiAB1C1 core oscillator only. Interaction with KaiC2 and KaiC3 proteins was not detected, suggesting different signal transduction components for the clock homologs. Inactivation of rpaA in Synechocystis sp. PCC 6803 leads to reduced viability of the mutant in light-dark cycles, especially under mixotrophic growth conditions. Chemoheterotrophic growth of the ∆rpaA strain in the dark was abolished completely. Transcriptomic data revealed that RpaA is mainly involved in the regulation of genes related to CO2 - acclimation in the light and to carbon metabolism in the dark. Further, our results indicate a link between the circadian clock and phototaxis.

The small regulatory RNA SyR1/PsrR1 controls photosynthetic functions in cyanobacteria.

Little is known so far about RNA regulators of photosynthesis in plants, algae, or cyanobacteria. The small RNA PsrR1 (formerly SyR1) has been discovered in Synechocystis sp PCC 6803 and appears to be widely conserved within the cyanobacterial phylum. Expression of PsrR1 is induced shortly after a shift from moderate to high-light conditions. Artificial overexpression of PsrR1 led to a bleaching phenotype under moderate light growth conditions. Advanced computational target prediction suggested that several photosynthesis-related mRNAs could be controlled by PsrR1, a finding supported by the results of transcriptome profiling experiments upon pulsed overexpression of this small RNA in Synechocystis sp PCC 6803. We confirmed the interaction between PsrR1 and the ribosome binding regions of the psaL, psaJ, chlN, and cpcA mRNAs by mutational analysis in a heterologous reporter system. Focusing on psaL as a specific target, we show that the psaL mRNA is processed by RNase E only in the presence of PsrR1. Furthermore, we provide evidence for a posttranscriptional regulation of psaL by PsrR1 in the wild type at various environmental conditions and analyzed the consequences of PsrR1-based regulation on photosystem I. In summary, computational and experimental data consistently establish the small RNA PsrR1 as a regulatory factor controlling photosynthetic functions.

Cell-Free Enzymatic Conversion of Spent Coffee Grounds Into the Platform Chemical Lactic Acid.

The coffee industry produces over 10 billion kg beans per year and generates high amounts of different waste products. Spent coffee grounds (SCG) are an industrially underutilized waste resource, which is rich in the polysaccharide galactomannan, a polysaccharide consisting of a mannose backbone with galactose side groups. Here, we present a cell-free reaction cascade for the conversion of mannose, the most abundant sugar in SCG, into L-lactic acid. The enzymatic conversion is based on a so far unknown oxidative mannose metabolism from Thermoplasma acidophilum and uses a previously characterized mannonate dehydratase to convert mannose into lactic acid via 4 enzymatic reactions. In comparison to known in vivo metabolisms the bioconversion is free of phosphorylated intermediates and cofactors. Assessment of enzymes, adjustment of enzyme loadings, substrate and cofactor concentrations, and buffer ionic strength allowed the identification of crucial reaction parameters and bottlenecks. Moreover, reactions with isotope labeled mannose enabled the monitoring of pathway intermediates and revealed a reverse flux in the conversion process. Finally, 4.4 ± 0.1 mM lactic acid was produced from 14.57 ± 0.7 mM SCG-derived mannose. While the conversion efficiency of the process can be further improved by enzyme engineering, the reaction demonstrates the first multi-enzyme cascade for the bioconversion of SCG.

Tools and strategies for constructing cell-free enzyme pathways.

Single enzyme systems or engineered microbial hosts have been used for decades but the notion of assembling multiple enzymes into cell-free synthetic pathways is a relatively new development. The extensive possibilities that stem from this synthetic concept makes it a fast growing and potentially high impact field for biomanufacturing fine and platform chemicals, pharmaceuticals and biofuels. However, the translation of individual single enzymatic reactions into cell-free multi-enzyme pathways is not trivial. In reality, the kinetics of an enzyme pathway can be very inadequate and the production of multiple enzymes can impose a great burden on the economics of the process. We examine here strategies for designing synthetic pathways and draw attention to the requirements of substrates, enzymes and cofactor regeneration systems for improving the effectiveness and sustainability of cell-free biocatalysis. In addition, we comment on methods for the immobilisation of members of a multi-enzyme pathway to enhance the viability of the system. Finally, we focus on the recent development of integrative tools such as in silico pathway modelling and high throughput flux analysis with the aim of reinforcing their indispensable role in the future of cell-free biocatalytic pathways for biomanufacturing.