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The most widely used isoprene emission algorithm, G93 formula, estimates instantaneous leaf-level isoprene emission using the basal emission factor and light and temperature dependency parameters. The G93 parameters have been suggested to show variation depending on past weather conditions, but no study has closely examined the relationship between past meteorological data and the algorithm parameters. Here, to examine the influence of the past weather on these parameters, we monitored weather conditions, G93 parameters, isoprene synthase transcripts and protein levels, and MEP pathway metabolites in the tropical tree Ficus septica for 12 days and analyzed their relationship with cumulative temperature and light intensity. Plants were illuminated with varying (ascending and descending) light regimes, and our previously developed Ping-Pong optimization method was used to parameterize G93. The cumulative temperature of the past 5 and 7 days positively correlated with CT2 and α, respectively, while the cumulative light intensity of the past 10 days showed the highest negative correlation with α. Concentrations of MEP pathway metabolites and IspS gene expression increased with increasing cumulative temperature. At best, the cumulative temperature of the past 2 days positively correlated with the MEP pathway metabolites and IspS gene expression, while these factors showed a biphasic positive and negative correlation with cumulative light intensity. Optimized G93 captured well the temperature and light dependency of isoprene emission at the beginning of the experiment; however, its performance significantly decreased for the latter stages of the experimental duration, especially for the descending phase. This was successfully improved through separate optimization of the ascending and descending phases, emphasizing the importance of the optimization of formula parameters and model improvement. These results have important implications for the improvement of isoprene emission algorithms, particularly under the predicted increase in future global temperatures.

Abstract It has been suggested that isoprene synthesis by isoprene synthase (IspS) proceeds via a substrate-assisted mechanism. The authors observed a non-enzymatic isoprene formation by Mn2+, which represents the basis of IspS enzyme reaction. Because IspS and many other terpene synthases require Mn2+ metal ions as cofactor, this study characterized the formation reaction for the first time. Metal ions including Mn2+ non-enzymatically produced both isoprene and 2-methyl-3-buten-2-ol (2-MBO) from dimethylallyl pyrophosphate (DMADP). Isoprene formation was most enhanced by Fe2+ and, to a lesser extent, by Mn2+ or Cu2+. Ni2+, Co2+, Mg2+, and Ba2+ exhibited a low activity to generate both isoprene and 2-MBO. The proportion of isoprene and 2-MBO varied with the Mn2+ concentration: isoprene predominated over 2-MBO at a higher Mn2+ concentration. Similarly, isoprene formation by Mn2+ increased exponentially as temperature increased with predominance of isoprene over 2-MBO at higher temperature. Both isoprene and 2-MBO formation was enhanced by acidic and neutral pH compared to alkaline conditions. Molecular dynamic simulation of DMADP suggested that the formation reaction is initiated by deprotonation of hydrogen on allyl terminal carbon by phosphate oxygen and generates carbocation and allyl anion intermediates. This is followed by quenching to produce isoprene or by hydroxyl addition to form 2-MBO. Thus, this study provided an insight into reaction mechanism of isoprene and 2-MBO biosynthesis and highlighted some parts of isoprene emission from terrestrial plants, which could be formed by non-enzymatic mechanism.

5-Deoxy(iso)flavonoids are structural representatives of phenylpropanoid-derived compounds and play critical roles in plant ecophysiology. Recently, 5-deoxy(iso)flavonoids gained significant interest due to their potential applications as pharmaceuticals, nutraceuticals, and food additives. Given the difficulties in their isolation from native plant sources, engineered biosynthesis of 5-deoxy(iso)flavonoids in a microbial host is a highly promising alternative approach. However, the production of 5-deoxy(iso)flavonoids is hindered by metabolic flux imbalances that result in a product profile predominated by non-reduced analogues. In this study, GmCHS7 (chalcone synthase from Glycine max) and GuCHR (chalcone reductase from Glycyrrhizza uralensis) were preliminarily utilized to improve the CHR ratio (CHR product to total CHS product). The use of this enzyme combination improved the final CHR ratio from 39.7% to 50.3%. For further optimization, a protein-protein interaction strategy was employed, basing on the spatial adhesion of GmCHS7:PDZ and GuCHR:PDZlig. This strategy further increased the ratio towards the CHR-derived product (54.7%), suggesting partial success of redirecting metabolic flux towards the reduced branch. To further increase the total carbon metabolic flux, 15 protein scaffolds were programmed with stoichiometric arrangement of the three sequential catalysts GmCHS7, GuCHR and MsCHI (chalcone isomerase from Medicago sativa), resulting in a 1.4-fold increase in total flavanone production, from 69.4 mg/L to 97.0 mg/L in shake flasks. The protein self-assembly strategy also improved the production and direction of the lineage-specific compounds 7,4′-dihydroxyflavone and daidzein in Escherichia coli. This study presents a significant advancement of 5-deoxy(iso)flavonoid production and provides the foundation for production of value-added 5-deoxy(iso)flavonoids in microbial hosts.

Engineering Taxol pathway in microbes needs to overcome the difficulty of expressing plant P450 enzymes. Here, the authors use a compartmentalized metabolic engineering strategy to construct the taxanes production pathway in chloroplasts of Nicotiana benthamiana and realize the production of taxadience-5α-ol.

The diterpenoid paclitaxel (Taxol®) is a blockbuster anticancer agent that was originally isolated from the Pacific yew (Taxus brevifolia) five decades ago. Despite the wealth of information gained over the years on Taxol research, there still remains supply issues to meet increasing clinical demand. Although alternative Taxol production methods have been developed, they still face several drawbacks that cause supply shortages and high production costs. It is highly desired to develop biotechnological production platforms for Taxol, however, there are still gaps in our understanding of the biosynthetic pathway, catalytic enzymes, regulatory and control mechanisms that hamper production of this critical drug by synthetic biology approaches. Over the past 5 years, significant advances were made in metabolic engineering and optimization of the Taxol pathway in different hosts, leading to accumulation of taxane intermediates. Computational and experimental approaches were leveraged to gain mechanistic insights into the catalytic cycle of pathway enzymes and guide rational protein engineering efforts to improve catalytic fitness and substrate/product specificity, especially of the cytochrome P450s (CYP450s). Notable breakthroughs were also realized in engineering the pathway in plant hosts that are more promising in addressing the challenging CYP450 chemistry. Here, we review these recent advances and in addition, we summarize recent transcriptomic data sets of Taxus species and elicited culture cells, and give a bird’s-eye view of the information that can be gleaned from these publicly available resources. Recent mining of transcriptome data sets led to discovery of two putative pathway enzymes, provided many lead candidates for the missing steps and provided new insights on the regulatory mechanisms governing Taxol biosynthesis. All these inferences are relevant to future biotechnological production of Taxol.

In this study, 2 types of solidified noncentrifugal brown sugars (W-NCS and P-NCS) were prepared from the whole stalk and separated pith, respectively, of raw sugarcane ( Saccharum officinarum L.). These products were discriminated in terms of their quality attributes, including color, sugars and minerals composition, taste, aroma, and antioxidant activity. The brown color of P-NCS was clearly different compared with that of W-NCS with a color difference value (Δ E*) of 9.36. There was no difference in the sugars and minerals composition between the 2 types of sugar, which led to very similar taste profiles. However, P-NCS had a weaker aroma intensity than W-NCS did. Moreover, P-NCS retained more than 60% of the antioxidant activity of W-NCS. The information gleaned from this study might be used to select appropriate end-uses for these 2 types of sugars. [ABSTRACT FROM AUTHOR]

Isoprene is the most abundant type of nonmethane, biogenic volatile organic compound in the atmosphere, and it is produced mainly by terrestrial plants. The tropical tree species Ficus septica Burm. F. (Rosales: Moraceae) has been shown to cease isoprene emissions when exposed to temperatures of 12 °C or lower and to re-induce isoprene synthesis upon subsequent exposure to temperatures of 30 °C or higher for 24 h. To elucidate the regulation of genes underlying the disabling and then induction of isoprene emission during acclimatization to ambient temperature, we conducted gene expression analyses of F. septica plants under changing temperature using quantitative real-time polymerase chain reaction and western blotting. Transcription levels were analyzed for 17 genes that are involved in metabolic pathways potentially associated with isoprene biosynthesis, including isoprene synthase (ispS). The protein levels of ispS were also measured. Changes in transcription and protein levels of the ispS gene, but not in the other assessed genes, showed identical temporal patterns to isoprene emission capacity under the changing temperature regime. The ispS protein levels strongly and positively correlated with isoprene emission capacity (R² = 0.92). These results suggest that transcriptional regulation of ispS gave rise to the temporal variation in isoprene emission capacity in response to changing temperature. [ABSTRACT FROM AUTHOR]