Mixing and matching methylotrophic enzymes to design a novel methanol utilization pathway in E. coli.

One-carbon (C1) compounds, such as methanol, have recently gained attention as alternative low-cost and non-food feedstocks for microbial bioprocesses. Considerable research efforts are thus currently focused on the generation of synthetic methylotrophs by transferring methanol assimilation pathways into established bacterial production hosts. In this study, we used an iterative combination of dry and wet approaches to design, implement and optimize this metabolic trait in the most common chassis, E. coli. Through in silico modelling, we designed a new route that "mixed and matched" two methylotrophic enzymes: a bacterial methanol dehydrogenase (Mdh) and a dihydroxyacetone synthase (Das) from yeast. To identify the best combination of enzymes to introduce into E. coli, we built a library of 266 pathway variants containing different combinations of Mdh and Das homologues and screened it using high-throughput 13C-labeling experiments. The highest level of incorporation of methanol into central metabolism intermediates (e.g. 22% into the PEP), was obtained using a variant composed of a Mdh from A. gerneri and a codon-optimized version of P. angusta Das. Finally, the activity of this new synthetic pathway was further improved by engineering strategic metabolic targets identified using omics and modelling approaches. The final synthetic strain had 1.5 to 5.9 times higher methanol assimilation in intracellular metabolites and proteinogenic amino acids than the starting strain did. Broadening the repertoire of methanol assimilation pathways is one step further toward synthetic methylotrophy in E. coli.

Combinatorial control by the protein kinases PKA, PHO85 and SNF1 of transcriptional induction of the Saccharomyces cerevisiae GSY2 gene at the diauxic shift.

Genes involved in storage carbohydrate metabolism are coordinately induced when yeast cells are subjected to conditions of stress, or when they exit the exponential growth phase on glucose. We show that the STress Responsive Elements (STREs) present in the promoter of GSY2 are essential for gene activation under conditions of stress, but dispensable for gene induction and glycogen accumulation at the diauxic shift on glucose. Using serial promoter deletion, we found that the latter induction could not be attributed to a single cis -regulatory sequence, and present evidence that this mechanism depends on combinatorial transcriptional control by signalling pathways involving the protein kinases Pho85, Snf1 and PKA. Two contiguous regions upstream of the GSY2 coding region are necessary for negative control by the cyclin-dependent protein kinase Pho85, one of which is a 14-bp G/C-rich sequence. Positive control by Snf1 is mediated by Mig1p, which acts indirectly on the distal part of the GSY2 promoter. The PKA pathway has the most pronounced effect on GSY2, since transcription of this gene is almost completely abolished in an ira1ira2 mutant strain in which PKA is hyperactive. The potent negative effect of PKA is dependent upon a branched pathway involving the transcription factors Msn2/Msn4p and Sok2p. The SOK2 branch was found to be effective only under conditions of high PKA activity, as in a ira1ira2 mutant, and this effect was independent of Msn2/4p. The Msn2/4p branch, on the other hand, positively controls GSY2 expression directly through the STREs, and indirectly via a factor that still remains to be discovered. In summary, this study shows that the transcription of GSY2 is regulated by several different signalling pathways which reflect the numerous factors that influence glycogen synthesis in yeast, and suggests that the PKA pathway must be deactivated to allow gene induction at the diauxic shift.

The Saccharomyces cerevisiae YPR184w gene encodes the glycogen debranching enzyme.

The YPR184w gene encodes a 1536-amino acid protein that is 34-39% identical to the mammal, Drosophila melanogaster and Caenorhabditis elegans glycogen debranching enzyme. The N-terminal part of the protein possesses the four conserved sequences of the alpha-amylase superfamily, while the C-terminal part displays 50% similarity with the C-terminal of other eukaryotic glycogen debranching enzymes. Reliable measurement of alpha-1,4-glucanotransferase and alpha-1, 6-glucosidase activity of the yeast debranching enzyme was determined in strains overexpressing YPR184w. The alpha-1, 4-glucanotransferase activity of a partially purified preparation of debranching enzyme preferentially transferred maltosyl units than maltotriosyl. Deletion of YPR184w prevents glycogen degradation, whereas overexpression had no effect on the rate of glycogen breakdown. In response to stress and growth conditions, the transcriptional control of YPR184w gene, renamed GDB1 (for Glycogen DeBranching gene), is strictly identical to that of other genes involved in glycogen metabolism.

STRE- and cAMP-independent transcriptional induction of Saccharomyces cerevisiae GSY2 encoding glycogen synthase during diauxic growth on glucose.

It has been shown that the so-called stationary phase GSY2 gene encoding glycogen synthase was induced as the cells left the exponential phase of growth, while glucose and all other nutrients were still plentiful in the medium (Parrou et al., 1999). Since this effect was essentially controlled at the transcriptional level, we looked for the cis- and trans-acting elements required for this specific growth-related genetic event. We demonstrated that mutations of the HAP2/3/4 binding site and of the two STress-Responsive cis-Elements (STRE) did not abolish the early induction of GSY2, although the latter mutation led to a 20-fold drop in the transcriptional activity of the promoter, as determined from lacZ gene fusions. Insertion of a DNA fragment (from -390 to -167 bp, relative to the ATG) of the promoter lacking the two STREs, upstream to the TATA box of a CYC1-lacZ fusion gene, allowed this reporter gene to be induced with a kinetic similar to that of GSY2-lacZ. Mutations in BCY1, which results in a hyperactive protein kinase A, did not alleviate the early induction, while causing a five- to 10-fold reduction in the transcriptional activity of GSY2. In addition, the repressive effect of protein kinase A was quantitatively conserved when both STREs were mutated in GSY2 promoter, indicating that the negative control of gene expression by the RAS-cAMP signalling pathway does not act solely through STREs. Taken together, these results are indicative of an active process that couples growth control to dynamic glucose consumption.

Dynamic responses of reserve carbohydrate metabolism under carbon and nitrogen limitations in Saccharomyces cerevisiae.

The dynamic responses of reserve carbohydrates with respect to shortage of either carbon or nitrogen source was studied to obtain a sound basis for further investigations devoted to the characterization of mechanisms by which the yeast Saccharomyces cerevisiae can cope with nutrient limitation during growth. This study was carried out in well-controlled bioreactors which allow accurate monitoring of growth and frequent sampling without disturbing the culture. Under glucose limitation, genes involved in glycogen and trehalose biosynthesis (GLG1, GSY1, GSY2, GAC1, GLC3, TPS1), in their degradation (GPH1, NTHI), and the typical stress-responsive CTT1 gene were coordinately induced in parallel with glycogen, when the growth has left the pure exponential phase and while glucose was still plentiful in the medium. Trehalose accumulation was delayed until the diauxic shift, although TPS1 was induced much earlier, due to hydrolysis of trehalose by high trehalase activity. In contrast, under nitrogen limitation, both glycogen and trehalose began to accumulate at the precise time when the nitrogen source was exhausted from the medium, coincidentally with the transcriptional activation of genes involved in their metabolism. While this response to nitrogen starvation was likely mediated by the stress-responsive elements (STREs) in the promoter of these genes, we found that these elements were not responsible for the co-induction of genes involved in reserve carbohydrate metabolism during glucose limitation, since GLG1, which does not contain any STRE, was coordinately induced with GSY2 and TPS1.