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The genetic stability and metabolic robustness of production strains is one of the key criteria for the production of bio-based products by microbial fermentation on an industrial scale. These criteria were here explored in an industrial ethanol-producer strain of Saccharomyces cerevisiae able to co-ferment D-xylose and L-arabinose with glucose through the chromosomal integration of several copies of pivotal genes for the use of these pentose (C5) sugars. Using batch sequential cultures in a controlled bioreactor that mimics long-term fermentation in an industrial setting, this strain was found to exhibit significant fluctuations in D-xylose and L-arabinose consumption as early as the 50th generation and beyond. These fluctuations seem not related to the few low-consumption C5 sugar clones that appeared throughout the sequential batch cultures at a frequency lower than 1.5% and that were due to the reduction in the number of copies of transgenes coding for C5 sugar assimilation enzymes. Also, subpopulations enriched with low or high RAD52 expression, whose expression level was reported to be proportional to homologous recombination rate did not exhibit defect in C5-sugar assimilation, arguing that other mechanisms may be responsible for copy number variation of transgenes. Overall, this work highlighted the existence of genetic and metabolic instabilities in an industrial yeast which, although modest in our conditions, could be more deleterious in harsher industrial conditions, leading to reduced production performance.

Rapeseed meal (RSM) is a major by-product of oil extraction from rapeseed, consists mainly of proteins and phenolic compounds. The use of RSM as protein feedstock for microbial fermentation is always hampered by phenolic compounds, which have antioxidant property with health-promoting benefits but inhibit bacterial growth. However, there is still not any good process that simultaneously improve extraction efficiency of phenolic compounds with conversion efficiency of protein residue into microbial production. Here we established a two-step strategy including fungal pretreatment followed by extraction of phenolic compounds. This could not only increase extraction efficiency and antioxidant property of phenolic compounds by about 2-fold, but also improve conversion efficiency of protein residue into iturin A production by Bacillus amyloliquefaciens CX-20 by about 33%. The antioxidant and antibacterial activities of phenolic extracts were influenced by both total phenolic content and profile, while microbial feedstock value of residue was greatly improved because protein content was increased by ∼5% and phenolic content was decreased by ∼60%. Moreover, this two-step process resulted in isolating more proteins from RSM, bringing iturin A production to 1.95 g/L. In conclusion, high-value-added and graded utilization of phenolic extract and protein residue from RSM with zero waste is realized by a two-step strategy, which combines both benefits of fungal pretreatment and phenolic extraction procedures.

The global warming conjugated with our reliance to petrol derived processes and products have raised strong concern about the future of our planet, asking urgently to find sustainable substitute solutions to decrease this reliance and annihilate this climate change mainly due to excess of CO2 emission. In this regard, the exploitation of microorganisms as microbial cell factories able to convert non-edible but renewable carbon sources into biofuels and commodity chemicals appears as an attractive solution. However, there is still a long way to go to make this solution economically viable and to introduce the use of microorganisms as one of the motor of the forthcoming bio-based economy. In this review, we address a scientific issue that must be challenged in order to improve the value of microbial organisms as cell factories. This issue is related to the capability of microbial systems to optimize carbon conservation during their metabolic processes. This initiative, which can be addressed nowadays using the advances in Synthetic Biology, should lead to an increase in products yield per carbon assimilated which is a key performance indice in biotechnological processes, as well as to indirectly contribute to a reduction of CO2 emission.

Glycolic acid is a two-carbon α-hydroxy acid with many applications in industrial sectors including packaging, fine chemistry, cosmetics, and pharmaceutics. Currently, glycolic acid is chemically manufactured from fossil resources. This chemical mode of production is raising some concerns regarding its use in health for personal care. Microbial production of GA stands as a remarkable challenge to meet these concerns, while responding to the increasing demand to produce bio-sourced products from renewable carbon resources. We here report on the design and expression of a novel non-natural pathway of glycolic acid in E. coli. The originality of this new pathway, termed “glycoptimus” relies on two pillars. On the one hand, it requires the overexpression of three naturally occurring E. coli genes, namely kdsD encoding a D-arabinose-5-P isomerase, fsaA encoding a class 1 aldolase that cleaves D-arabinose-5-P into glyceraldehyde-3-P and glycolaldehyde, and aldA coding for an aldehyde dehydrogenase that oxidizes glycoladehyde in glycolate. These three genes constitute the “glycoptimus module.” On the other hand, the expression of these genes together with a reshaping of the central carbon metabolism should enable a production of glycolic acid from pentose and hexose at a molar ratio of 2.5 and 3, respectively, which corresponds to 50% increase as compared to the existing pathways. We demonstrated the ‘in vivo’ potentiality of this pathway using an E. coli strain, which constitutively expressed the glycoptimus module and whose carbon flow in glycolysis was blocked at the level of glyceraldehyde-3-P dehydrogenase reaction step. This engineered strain was cultivated on a permissive medium containing malate and D-glucose. Upon exhaustion of malate, addition of either D-glucose, D-xylose or L-arabinose led to the production of glycolic acid reaching about 30% of the maximum molar yield. Further improvements at the level of enzymes, strains and bioprocess engineering are awaited to increase yield and titer, rendering the microbial production of glycolic acid affordable for a cost-effective industrial process.