Adipic acid is a platform chemical, and is the most important commercial dicarboxylic acid. It has been targeted for biochemical conversion as an alternative to present chemical production routes. From the perspective of bioeconomy, several kinds of raw material are of interest including the sugar platform (derived from starch, cellulose or hemicellulose), the lignin platform (aromatics) and the fatty acid platform (lipid derived). Two main biochemical-based production schemes may be employed: (i) direct fermentation to adipic acid, or (ii) fermentation to muconic or glucaric acid, followed by chemical hydrogenation (indirect fermentation). This review presents a comprehensive description of the metabolic pathways that could be constructed and analyzes their respective theoretical yields and metabolic constraints. The experimental yields and titers obtained so far are low, with the exception of processes based on palm oil and glycerol, which have been reported to yield up to 50 g and 68 g adipic acid/L, respectively. The challenges that remain to be addressed in order to achieve industrially relevant production levels include solving redox constraints, and identifying and/or engineering enzymes for parts of the metabolic pathways that have yet to be metabolically demonstrated. This review provides new insights into ways in which metabolic pathways can be constructed to achieve efficient adipic acid production. The production host provides the chassis to be engineered via an appropriate metabolic pathway, and should also have properties suitable for the industrial production of adipic acid. An acidic process pH is attractive to reduce the cost of downstream processing. The production host should exhibit high tolerance to complex raw material streams and high adipic acid concentrations at acidic pH.

We investigated the severity of the inhibitory effects of 13 phenolic compounds usually found in spruce hydrolysates (4-hydroxy-3-methoxycinnamaldehyde, homovanilyl alcohol, vanillin, syringic acid, vanillic acid, gallic acid, dihydroferulic acid,p-coumaric acid, hydroquinone, ferulic acid, homovanillic acid, 4-hydroxybenzoic acid and vanillylidenacetone). The effects of the selected compounds on cell growth, biomass yield and ethanol yield were studied and the toxic concentration threshold was defined for each compound. Using Ethanol Red, the popular industrial strain ofSaccharomyces cerevisiae, we found the most toxic compound to be 4-hydroxy-3-methoxycinnamaldehyde which inhibited growth at a concentration of 1.8 mM. We also observed that toxicity did not generally follow a trend based on the aldehyde, acid, ketone or alcohol classification of phenolic compounds, but rather that other structural properties such as additional functional groups attached to the compound may determine its toxicity. Three distinctive growth patterns that effectively clustered all the compounds involved in the screening into three categories. We suggest that the compounds have different cellular targets, and that. We suggest that the compounds have different cellular targets and inhibitory mechanisms in the cells, also compounds who share similar pattern on cell growth may have similar inhibitory effect and mechanisms of inhibition.

Background: Pretreatment of biomass for lignocellulosic ethanol production generates compounds that can inhibit microbial metabolism. The furan aldehydes hydroxymethylfurfural (HMF) and furfural have received increasing attention recently. In the present study, the effects of HMF and furfural on redox metabolism, energy metabolism and gene expression were investigated in anaerobic chemostats where the inhibitors were added to the feed-medium. Results: By cultivating the xylose-utilizing Saccharomyces cerevisiae strain VTT C-10883 in the presence of HMF and furfural, it was found that the intracellular concentrations of the redox co-factors and the catabolic and anabolic reduction charges were significantly lower in the presence of furan aldehydes than in cultivations without inhibitors. The catabolic reduction charge decreased from 0.13(±0.005) to 0.08(±0.002) and the anabolic reduction charge decreased from 0.46(±0.11) to 0.27(±0.02) when HMF and furfural were present. The intracellular ATP concentration was lower when inhibitors were added, but resulted only in a modest decrease in the energy charge from 0.87(±0.002) to 0.85(±0.004) compared to the control. Transcriptome profiling followed by MIPS functional enrichment analysis of up-regulated genes revealed that the functional group “Cell rescue, defense and virulence” was over-represented when inhibitors were present compared to control cultivations. Among these, the ATP-binding efflux pumps PDR5 and YOR1 were identified as important for inhibitor efflux and possibly a reason for the lower intracellular ATP concentration in stressed cells. It was also found that genes involved in pseudohyphal growth were among the most up-regulated when inhibitors were present in the feed-medium suggesting nitrogen starvation. Genes involved in amino acid metabolism, glyoxylate cycle, electron transport and amino acid transport were enriched in the down-regulated gene set in response to HMF and furfural. It was hypothesized that the HMF and furfural-induced NADPH drainage could influence ammonia assimilation and thereby give rise to the nitrogen starvation response in the form of pseudohyphal growth and down-regulation of amino acid synthesis. Conclusions: The redox metabolism was severely affected by HMF and furfural while the effects on energy metabolism were less evident, suggesting that engineering of the redox system represents a possible strategy to develop more robust strains for bioethanol production.

Background: One of the crucial factors for a sustainable and economical production of lignocellulosic based bioethanol is the availability of a robust fermenting microorganism with high tolerance to inhibitors generated during the pretreatment of lignocellulosic raw materials, since these inhibitors are known to severely hinder growth and fermentation. Results: A long-term adaptation in repetitive batch cultures in shake flasks using a cocktail of 12 different inhibitors and a long-term chemostat adaptation using spruce hydrolysate were used as evolutionary engineering strategies to improve the inhibitor tolerance in the metabolically engineered xylose utilizing Saccharomyces cerevisiae strain, TMB3400. The yeast was evolved for a period of 429 and 97 generations in repetitive batch cultures and chemostat cultivation, respectively. During the evolutionary engineering in repetitive batch cultures the maximum specific growth rate increased from 0.18 h^-1 to 0.33 h^-1 and the time of lag phase was decreased from 48 h to 24 h. In the chemostat adaptation, after 97 generations, the specific conversion rates of HMF and furfural were found to be 3.5 and 4 folds higher respectively, compared to rates after three generations. Two evolved strains (RK60-5, RKU90-3) and one evolved strain (KE1-17) were isolated from evolutionary engineering in repetitive batches and chemostat cultivation, respectively. The strains displayed significantly improved growth performance over TMB3400 when cultivated in spruce hydrolysate under anaerobic conditions, the evolved strains exhibited 25 to 38% increase in specific consumption rate of sugars and 32 to 50% increased specific ethanol productivity compared to TMB3400. The evolved strains RK60-5 and RKU90-3 were unable to consume xylose under anaerobic conditions, whereas, KE1-17 was found to consume xylose at similar rates as TMB3400. Conclusion: Using evolutionary engineering strategies in batch and chemostat cultivations we have generated three evolved strains that show significantly better tolerance to inhibitors in spruce hydrolysate and displayed a shorter time for overall fermentation of sugars compared to the parental strain.