No abstract

Aliphatic hydrocarbons such as fatty alcohols and petroleum-derived alkanes have numerous applications in the chemical industry. In recent years, the renewable synthesis of aliphatic hydrocarbons has been made possible by engineering microbes to overaccumulate fatty acids. However, to generate end products with the desired physicochemical properties (e.g., fatty aldehydes, alkanes, and alcohols), further conversion of the fatty acid is necessary. A carboxylic acid reductase (CAR) from Mycobacterium marinum was found to convert a wide range of aliphatic fatty acids (C 6 –C 18 ) into corresponding aldehydes. Together with the broad-substrate specificity of an aldehyde reductase or an aldehyde decarbonylase, the catalytic conversion of fatty acids to fatty alcohols (C 8 –C 16 ) or fatty alkanes (C 7 –C 15 ) was reconstituted in vitro. This concept was applied in vivo , in combination with a chain-length-specific thioesterase, to engineer Escherichia coli BL21(DE3) strains that were capable of synthesizing fatty alcohols and alkanes. A fatty alcohol titer exceeding 350 mg·L −1 was obtained in minimal media supplemented with glucose. Moreover, by combining the CAR-dependent pathway with an exogenous fatty acid-generating lipase, natural oils (coconut oil, palm oil, and algal oil bodies) were enzymatically converted into fatty alcohols across a broad chain-length range (C 8 –C 18 ). Together with complementing enzymes, the broad substrate specificity and kinetic characteristics of CAR opens the road for direct and tailored enzyme-catalyzed conversion of lipids into user-ready chemical commodities.

Flavonols constitute a major class of plant natural products (PNPs) 1 that share a common flavonoid nucleus and that accumulate in a wide range of conjugate structures. For example, over 350 different conjugate forms of a single flavonol, quercetin, have been observed to accumulate in plants to date (1). A large proportion of this structural variability is due to the attachment of one or several sugar moieties at different positions as illustrated in Fig.

Plants are exposed to a wide range of toxic and bioactive low-molecular-weight molecules from both exogenous and endogenous sources. Glycosylation is one of the primary sedative mechanisms that plants utilise in order to maintain metabolic homeostasis. Recently, a range of glycosyltransferases has been characterized in detail with regard to substrate specificity. The next step in increasing our understanding of the biology of glycosylation will require information regarding the exact role of individual glycosyltransferases in planta, as well as an insight into their potential involvement in metabolon-complexes. Hopefully, this will answer how a large number of glycosyltransferases with broad, rather than narrow, substrate specificity can be constrained in order to avoid interfering with other pathways of primary and secondary metabolism. These and other topics are discussed.

A precipitation assay is presented that enables tannin measurement in matrices of red wine, 50% ethanol grape extract and aqueous tannin solutions. By exploiting the polysaccharide polymer methyl cellulose to precipitate tannins, the absorbance of phenolics at 280 nm before and after tannin precipitation (subtractive approach) can be obtained, thus enabling selective measurement of tannin only. This methyl cellulose precipitable (MCP) tannin assay allows complete precipitation of tannin from red wine and from grape homogenate extracts. The subtractive assay is both simple and robust, selective for condensed tannins and does not suffer interference from other 280 nm‐absorbing phenolics such as anthocyanins or catechins. Matrix effects have only minimal impact on the assay performance and validation parameters indicate a robust performance. There was good correlation between tannin measured by reverse‐phase HPLC and the MCP tannin assay for 121 Australian red wines (r= 0.74) and also 54 grape extracts (r= 0.79). We envisage that the technical simplicity of this tannin assay will enable widespread research and field applications. In addition, an alternative format that requires re‐solubilisation of the tannin‐polymer pellet in acetonitrile is reported, which is particularly suitable for measurement of smaller tannin concentrations. Notwithstanding that option, technical requirements of the re‐solubilisation step lead us to suggest that the subtractive format would be simple for adoption by wine industry practitioners.

Slow denaturation of wine proteins is thought to lead to protein aggregation, flocculation into a hazy suspension and formation of precipitates. The majority of wine proteins responsible for haze are grape‐derived, have low isoelectric points and molecular weight. They are grape pathogenesis‐related (PR) proteins that are expressed throughout the ripening period post véraison, and are highly resistant to low pH and enzymatic or non‐enzymatic proteolysis. Protein levels in un‐fined white wine differ by variety and range up to 300 mg/L. Infection with some common grapevine pathogens or skin contact, such as occurs during transport of mechanically harvested fruit, results in enhanced concentrations of some PR proteins in juice and wine. Oenological control of protein instability is achieved through adsorption of wine proteins onto bentonite. The adsorption of proteins onto bentonite occurs within several minutes, suggesting that a continuous contacting process could be developed. The addition of proteolytic enzyme during short term heat exposure, to induce PR protein denaturation, showed promise as an alternative to bentonite fining. The addition of haze‐protective factors, yeast mannoproteins, to wines results in decreased particle size of haze, probably by competition with wine proteins for other non‐proteinaceous wine components required for the formation of large insoluble aggregations of protein. Other wine components likely to influence haze formation are ethanol concentration, pH, metal ions and phenolic compounds.

The ethylene-forming enzyme (EFE) from Pseudomonas syringae catalyzes the synthesis of ethylene which can be easily detected in the headspace of closed cultures. A synthetic codon-optimized gene encoding N-terminal His-tagged EFE (EFEh) was expressed in Synechocystis sp. PCC 6803 (Synechocystis) and Escherichia coli (E. coli) under the control of diverse promoters in a self-replicating broad host-range plasmid. Ethylene synthesis was stably maintained in both organisms in contrast to earlier work in Synechococcus elongatus PCC 7942. The rate of ethylene accumulation was used as a reporter for protein expression in order to assess promoter strength and inducibility with the different expression systems. Several metal-inducible cyanobacterial promoters did not function in E. coli but were well-regulated in cyanobacteria, albeit at a low level of expression. The E. coli promoter Ptrc resulted in constitutive expression in cyanobacteria regardless of whether IPTG was added or not. In contrast, a Lac promoter variant, PA1lacO-1, induced EFE-expression in Synechocystis at a level of expression as high as the Trc promoter and allowed a fine level of IPTG-dependent regulation of protein-expression. The regulation was tight at low cell density and became more relaxed in more dense cultures. A synthetic quorum-sensing promoter system was also constructed and shown to function well in E. coli, however, only a very low level of EFE-activity was observed in Synechocystis, independent of cell density.

The entire pathway for synthesis of the tyrosine-derived cyanogenic glucoside dhurrin has been transferred from Sorghum bicolor to Arabidopsis thaliana. Here, we document that genetically engineered plants are able to synthesize and store large amounts of new natural products. The presence of dhurrin in the transgenic A. thaliana plants confers resistance to the flea beetle Phyllotreta nemorum, which is a natural pest of other members of the crucifer group, demonstrating the potential utility of cyanogenic glucosides in plant defense.