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Delphastus catalinae (Horn) is a predatory ladybird beetle (Coccinellidae) commonly used as a biocontrol agent against greenhouse infestation by whiteflies. It belongs to the basal subfamily Microweisinae, a group for which chemical defenses have not been previously investigated. The larval and pupal stages of D. catalinae possess minute secretory hairs that produce droplets containing compounds of both isoprenoid and polyketide origin. Bioassays with the predatory ant Crematogaster lineolata showed both the larval and pupal secretions to be deterrent. Moreover, isolated secretion components, from both classes of compounds, displayed antipredator activity against the ant. Experiments with D. catalinae larvae fed isotopically labeled glucose showed C-incorporation into both categories of compounds within the pupal secretion, demonstrating that these antipredator compounds, which differ from the typical nitrogenous defensive molecules of coccinellids, are biosynthesized endogenously. This suggests that the wide use of alkaloids by more derived coccinellids may have arisen after their divergence from the more basal Microweisinae. [ABSTRACT FROM AUTHOR]

Cytochromes P450 (CYPs) belong to the superfamily of heme b containing monooxygenases with currently more than 21,000 members. These enzymes accept a vast range of organic molecules and catalyze diverse reactions. These extraordinary capabilities of CYP systems that are unmet by other enzymes make them attractive for biotechnology. However, the complexity of these systems due to the need of electron transfer from nicotinamide adenine dinucleotide (phosphate) (NAD(P)H) via redox partner proteins for the initial hydroxylation step limits a broader technical implementation of CYP enzymes. There have been several reviews during the past years tackling the potential CYPs for synthetic application. The aim of this review is to give a critical overview about possibilities and chances for application of these interesting catalysts as well as to discuss drawbacks and problems related to their use. Solutions to overcome these limitations will be demonstrated, and several selected examples of successful CYP applications under industrial conditions will be reviewed. [ABSTRACT FROM AUTHOR]

Abstract: De novo biosynthesis of linoleic acid (LA) and its conversion to (Z,Z)-6,9-heptadecadiene were examined in Carpoglyphus lactis (Acarina, Carpoglyphidae). Experiments involving 13C-administration using [1-13C]-d-glucose revealed that 13C atoms were incorporated into LA of total lipid extracted from the mite, resulting in labeling of all even-numbered carbons. This result demonstrated that LA was produced from 13C-labeled acetyl-CoA, which is indicative of direct de novo biosynthesis. In these feeding experiments involving [1-13C]-d-glucose, 13C atoms were also incorporated into (Z,Z)-6,9-heptadecadiene, which is one of the major secretory components in the mite. The labeling pattern of (Z,Z)-6,9-heptadecadiene at odd-numbered carbons agreed well with that of LA after loss of the carboxyl carbon. It was concluded that the mites could stably convert LA into (Z,Z)-6,9-heptadecadiene without the dietary requirement of this essential fatty acid. [Copyright &y& Elsevier]

Isobavachalcone (IBC) is a prenylated chalcone mainly distributed in some Fabaceae and Moraceae species. IBC exhibits a wide range of pharmacological properties, including anti-bacterial, anti-viral, anti-inflammatory, and anti-cancer activities. In this study, we attempted to construct the heterologous biosynthesis pathway of IBC in tobacco ( Nicotiana tabacum ). Four previously reported prenyltransferases, including GuILDT from Glycyrrhiza uralensis , HlPT1 from Humulus lupulus , and SfILDT and SfFPT from Sophora flavescens , were subjected to an in planta screening to verify their activities for the biosynthesis of IBC, by using tobacco transient expression with exogenous isoliquiritigenin as the substrate. Only SfFPT and HlPT1 could convert isoliquiritigenin to IBC, and the activity of SfFPT was higher than that of HlPT1. By co-expression of GmCHS8 and GmCHR5 from Glycine max , endogenous isoliquiritigenin was generated in tobacco leaves (21.0 μg/g dry weight). After transformation with a multigene vector carrying GmCHS8 , GmCHR5 , and SfFPT , de novo biosynthesis of IBC was achieved in transgenic tobacco T 0 lines, in which the highest amount of IBC was 0.56 μg/g dry weight. The yield of IBC in transgenic plants was nearly equal to that in SfFPT transient expression experiments, in which substrate supplement was sufficient, indicating that low IBC yield was not attributed to the substrate supplement. Our research provided a prospect to produce valuable prenylflavonoids using plant-based metabolic engineering., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Guo, Zhao, Wang, Yang, Wei, Guo, Dai, Hirai, Bao, Yang, Chen and Li.)

Anthocyanins are natural pigments found in various plants. As multifunctional natural compounds, anthocyanins are widely used in food, pharmaceuticals, health products, and cosmetics. At present, the anthocyanins are heterologously biosynthesized in prokaryotes from flavan-3-ols, which is rather expensive. This study aimed to metabolically engineer Saccharomyces cerevisiae for anthocyanin production. Anthocyanin production has been extensively studied to understand the metabolic pathway enzymes in their natural hosts, including CHS (chalcone synthase); FLS (flavonol synthase); CHI (chalcone isomerase); F3H (flavanone 3-hydroxylase); F3'H (flavonoid 3'-hydroxylase); F3'5'H (flavonoid 3',5'-hydroxylase); DFR (dihydroflavonol 4-reductase); ANS (anthocyanidin synthase); LAR (leucoanthocyanidin reductase); and UFGT (flavonoid 3-O-glucosyltransferase). The anthocyanin transporter Md GSTF6 was first introduced and proven to be indispensable for the biosynthesis of anthocyanins. By expressing Md GSTF6, Fa DFR, Ph ANS 0 , and Dc 3GT and disrupting EXG1 (the main anthocyanin-degrading enzyme), the BA-22 strain produced 261.6 mg/L (254.5 mg/L cyanidin-3- O -glucoside and 7.1 mg/L delphinidin-3- O -glucoside) anthocyanins from 2.0 g/L dihydroflavonols, which was known to be the highest titer in eukaryotes. Finally, 15.1 mg/L anthocyanins was obtained from glucose by expressing the de novo biosynthesis pathway in S. cerevisiae , which is known to be the highest de novo production. It is the first study to show that through the introduction of a plant anthocyanin transporter and knockout of a yeast endogenous anthocyanin degrading enzyme, the anthocyanin titer has been increased by more than 100 times., Competing Interests: Authors SX and GC were employed by the company Zhejiang Esigma Biotechnology Co., Ltd. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Xu, Li, Zhou, Chen and Shao.)

Abstract: The starch-synthase enzymes used in this study were the second acetone precipitate and Fractions 21 and 23, , [Mukerjea, Ru.; Falconer, D. J.; Yoon, S.-H.; Robyt, J. F. Carbohydr. Res. 2010, 345, 1555–1563]. Fractions 21 and 23 had high specific activities of 544 and 944 International Units/mg, respectively. When the enzymes and buffer and substrate were treated with immobilized α-amylase and glucoamylase for 30min, they all had the same activity, before and after treatment, indicating that the enzymes were free of putative primers and synthesized amylose chains de novo, without the addition of primers. Starch-synthase was immobilized and reacted with ADP-[14C]Glc; the immobilized enzyme was removed, washed and treated at pH 2 and 50°C for 30min, giving the release of 14C-d-glucopyranose and 14C-amylose, showing that during catalysis they were covalently attached to the enzyme active-site. Pulse and chase reactions of starch-synthase with ADP-[14C]Glc and ADPGlc, respectively, followed by reduction and acid hydrolysis of the starch-chain product, gave 14C-d-glucitol from the pulse reaction and a significant decrease of 14C-d-glucitol from the chase reaction, showing that the addition of d-glucose from ADPGlc was to the reducing-ends of the growing amylose chains. Reactions of four different concentrations of starch-synthase, with constant ADPGlc concentration and temperature, gave four amylose chains, each with different number average molecular weights that were inversely proportional to the concentration of the enzyme, indicating that the synthesis was processive. From the results, a two catalytic-site, insertion mechanism is proposed for the biosynthesis of starch chains. [Copyright &y& Elsevier]

Abstract: Structural diversity of polypropionates arises from the amazing combination of acetate and propionate units. Feeding experiments with single and doubly 13 C-labelled precursors prove that the Mediterranean slug Placida dendritica utilizes intact C3-units for the biosynthesis of placidenes (e.g., 1–5), prototypes of a family of pyrone-containing polypropionates largely represented in fungi and marine invertebrates. These results show that fungi and molluscs have elaborated two distinct polyketide pathways for the synthesis of similar or even identical compounds. [Copyright &y& Elsevier]

Abstract: Pyrimidine nucleotide metabolism was studied in tobacco callus cultured for 21days under shoot-forming (SF) and non-shoot-forming (NSF) conditions by following the metabolic fate of orotic acid, a precursor of the de novo pathway, and uridine and uracil, intermediates of the salvage and degradation pathways respectively. Nucleic acid synthesis was also investigated by measuring the incorporation of labeled thymidine into different cellular components. Our results indicate that with respect to nucleotide metabolism, the organogenic process in tobacco can be divided in two “metabolic phases”: a de novo phase followed by a salvage phase. The initial stages of meristemoid formation during tobacco organogenesis (up to day 8) are characterized by a heavy utilization of orotic acid into nucleotides and nucleic acids. Utilization of this intermediate for the de novo synthesis of nucleotides, which is limited in NSF tissue, is mainly due to the activity of orotate phosphoribosyltransferase (OPRT), which increases in tissue cultured under SF conditions. After day 8, nucleotide synthesis during shoot growth seems to be mainly due to the salvage activity of both uridine and uracil. Both intermediates are preferentially utilized in SF tissue for the formation of nucleotides and nucleic acids through the activities of their respective salvage enzymes: uridine kinase (URK), and uracil phosphoribosyltransferase (UPRT). Metabolic studies on thymidine indicate that in SF tissue maximal nucleic acid synthesis occurs at day 4, in support of the initiation of meristemoid formation. Overall these results suggest that the organogenic process in tobacco is underlined by precise fluctuations in pyrimidine metabolism which delineate structural events culminating in shoot formation. [Copyright &y& Elsevier]

Summary: Changes in the pattern of pyrimidine nucleotide metabolism were investigated in Pinus radiata cotyledons cultured under shoot-forming (SF; +N6-benzyladenine) and non-shoot-forming (NSF, −N6-benzyladenine) conditions, as well as in cotyledons unresponsive (OLD) to N6-benzyladenine. This was carried out by following the metabolic fate of externally supplied 14C-labeled orotic acid, intermediate of the de novo pathway, and 14C-labeled uridine and uracil, substrates of the salvage pathway. Nucleic acid synthesis was also investigated by following the metabolic fate of 14C-labeled thymidine during shoot bud formation and development. The de novo synthesis of pyrimidine nucleotides was operative under both SF and NSF conditions, and the activity of orotate phosphoribosyltransferase (OPRT), a key enzyme of the de novo pathway, was higher in SF tissue. Utilization of both uridine and uracil for nucleotide and nucleic acid synthesis clearly indicated that the salvage pathway of pyrimidine metabolism is also operative during shoot organogenesis. In general, uridine was a better substrate for the synthesis of salvage products than uracil, possibly due to the higher activity of uridine kinase (UK), compared to uracil phosphoribosyltransferase (UPRT). Incorporation of uridine into the nucleic acid fraction of OLD cotyledons was lower than that observed for their responsive (day 0) counterparts. Similarly, uracil utilization for nucleic acid synthesis was lower in NSF cotyledons, compared to that observed for SF tissue after 10 days in culture. This difference was ascribed to higher UPRT activity measured in the latter. Thus, there was an apparent difference in the utilization of nucleotides derived from uracil and uridine for nucleotide synthesis. The increased ability to produce pyrimidine nucleotides via the salvage pathway during shoot bud formation may be required in support of nucleic acid synthesis occurring during the process. Studies on thymidine metabolism confirmed this notion. [Copyright &y& Elsevier]

Abstract: The present results were obtained in the context of an attempt to develop an in vitro test intended to assess the genotoxicity of seawater. It is based on a short-term culture method of mussel mantle explants that gives mitotic cells in which DNA damage is likely to be detected. Its principle consists of the incorporation of the seawater to be tested in the culture medium. Two factors that influence cell proliferation were studied: (1) salinity of seawater and (2) basic composition of the culture medium i.e. replacement of Eagle’s Basal Medium (BME) by Eagle’s Minimum Essential Medium (MEM). When culture medium contained BME, a saliny change from 35‰ to 28‰ (that is the case for coastal or estuarine waters) resulted in a significant reduction of cell proliferation. In contrast, when culture medium was prepared with MEM, the same decrease of salinity did not change significantly the number of metaphase cells obtained. This suggested medium prepared with MEM allowed tissues in culture to better withstand the hypoosmotic shock generated by 28‰ seawater. This has been attributed to amino acids the concentration of which being higher in MEM than in BME. Direct and indirect mechanisms of osmoregulation are suggested to explain the observations. These results show that the test proposed could be used to assess the genotoxicity of coastal or estuarine seawater with salinity comprised from 28‰ to 35‰, provided that MEM is used. [Copyright &y& Elsevier]

Gas chromatography (GC) analyses of whole animal skin extracts and individual tissue extracts obtained from specimens of Cadlina luteomarginata collected in British Columbia and southern California were used to determine if concentrations of the nudibranch's biosynthetic products—albicanyl acetate ( 1), cadlinaldehyde ( 2), and luteone ( 3)—vary significantly between two populations, among individuals of a population, and among body tissues of individual specimens. The major biosynthetic product, albicanyl acetate ( 1), has the same concentration in both British Columbia and California populations, while the British Columbia population contains greater total amounts of 2 and 3 than the California population. Within individuals from one population, the largest proportion of endogenous metabolites is in the dorsum, specifically in the mantle dermal formations and margins. The GC analyses show that across geographically separated populations and within geographically localized populations the concentration of endogenous metabolites is inversely correlated with availability of structurally similar compounds from dietary sources. This suggests that the de novo biosynthesis of defensive compounds might be regulated according to need. [ABSTRACT FROM AUTHOR]

Salvianolic acid B (SAB), also named lithospermic acid B, belongs to a class of water-soluble phenolic acids, originating from plants such as Salvia miltiorrhiza . SAB exhibits a variety of biological activities and has been clinically used to treat cardio- and cerebrovascular diseases and also has great potential as a health care product and medicine for other disorders. However, its biosynthetic pathway has not been completely elucidated. Here, we report the de novo biosynthesis of SAB in Saccharomyces cerevisiae engineered with the heterologous rosmarinic acid (RA) biosynthetic pathway. The created pathway contains seven genes divided into three modules on separate plasmids, pRS424-FjTAL-Sm4CL2, pRS425-SmTAT-SmHPPR or pRS425-SmTAT-CbHPPR, and pRS426-SmRAS-CbCYP-CbCPR. These three modules were cotransformed into S. cerevisiae , resulting in the recombinant strains YW-44 and YW-45. Incubation of the recombinant strains in a basic medium without supplementing any substrates yielded 34 and 30 μg/L of SAB. The findings in this study indicate that the created heterologous RA pathway cooperates with the native metabolism of S. cerevisiae to enable the de novo biosynthesis of SAB. This provides a novel insight into a biosynthesis mechanism of SAB and also lays the foundation for the production of SAB using microbial cell factories.

Chlorogenic acid (CGA), a major dietary phenolic compound, has been increasingly used in the food and pharmaceutical industries because of its ready availability and extensive biological and pharmacological activities. Traditionally, extraction from plants has been the main approach for the commercial production of CGA. This study reports the first efficient microbial production of CGA by engineering the yeast, Saccharomyces cerevisiae , on a simple mineral medium. First, an optimized de novo biosynthetic pathway for CGA was reconstructed in S. cerevisiae from glucose with a CGA titer of 36.6 ± 2.4 mg/L. Then, a multimodule engineering strategy was employed to improve CGA production: (1) unlocking the shikimate pathway and optimizing carbon distribution; (2) optimizing the l-Phe branch and pathway balancing; and (3) increasing the copy number of CGA pathway genes. The combination of these interventions resulted in an about 6.4-fold improvement of CGA titer up to 234.8 ± 11.1 mg/L in shake flask cultures. CGA titers of 806.8 ± 1.7 mg/L were achieved in a 1 L fed-batch fermenter. This study opens a route to effectively produce CGA from glucose in S. cerevisiae and establishes a platform for the biosynthesis of CGA-derived value-added metabolites.

2-Acetamidophenol (AAP) is an aromatic product with promising activities in agricultural applications and medical research. At present, AAP is synthesized by chemical methods from nonrenewable fossil fuel resources, which cause environmental pollution and the reaction conditions are harsh. In this study, we constructed the artificial biosynthetic pathway of AAP with five different expressed proteins in Escherichia coli for the first time. By introducing the hydrogen peroxide degrading enzyme catalase and improving cell tolerance to toxic intermediates or products, the yield of AAP reached 33.54 mg/L using shaking-flask culture. The best-engineered strain could produce 568.57 mg/L AAP by fed-batch fermentation from glucose and precursor (2-aminophenol, 2-AP) addition. Furthermore, a one-pot whole-cell cascade biocatalytic pathway to AAP and analogues was developed and optimized. This method can efficiently produce 1.8 g/L AAP using the methyl anthranilate hydrolysis product as the substrate. This study provides not only the de novo artificial biosynthetic pathway of AAP in E. coli but also a promising sustainable and efficient strategy to enable the synthesis of AAP on a gram scale.

Zingerone (vanillylacetone; 4-hydroxy-3-methoxyphenylethyl methyl ketone) is a key component responsible for the pungency of ginger ( Zingiber officinale ). In this study, it was confirmed that a type III polyketide synthase (PKS) gene ( pmpks ) from Piper methysticum exhibits feruloyl-CoA-preferred benzalacetone synthase (BAS) activity. Based on these results, we constructed an artificial biosynthetic pathway for zingerone production from supplemented ferulic acid with 4-coumarate CoA ligase (4CL), PmPKS, and benzalacetone reductase (BAR). Furthermore, a de novo pathway for the production of zingerone was assembled using six heterologous genes, encoding tyrosine ammonia-lyase ( optal ), cinnamate-4-hydroxlase ( sam 5), caffeic acid O-methyltransferase ( com ), 4CL (4 cl 2 nt ), BAS ( pmpks ), and BAR ( rzs 1), in Escherichia coli . Using the engineered l-tyrosine-overproducing E. coli ΔCOS4 strain as a host, a maximum yield of 24.03 ± 2.53 mg/L zingerone was achieved by complete de novo synthesis.

• Special cultivation conditions were developed to address hydroxyhydroquinone stability issue. • An artificial pathway was designed and established for the hydroxyhydroquinone biosynthesis. • Modular co-culture engineering strategies were adopted to produce 64 mg/L hydroxyhydroquinone from 5 g/L glucose. Hydroxyhydroquinone (HHQ) is an aromatic triol with important biological activities and values. In this work, we achieved de novo HHQ bioproduction using glucose as the substrate. First, we established a cultivation method to address the issue of HHQ degradation in an aqueous solution. Second, an artificial biosynthesis pathway was constituted in E. coli to enable the conversion of glucose to HHQ via 4-hydroxybenzoate. Moreover, microbial co-culture engineering techniques were utilized to modularize the biosynthesis pathway and improve the HHQ production to 64 mg/L within 48 h. To our knowledge, this is the first report of heterologous biosynthesis of HHQ using a microbial host. The findings of this work lay a solid foundation for future efforts in microbial bioproduction of HHQ and related biochemicals. [ABSTRACT FROM AUTHOR]

Sialic acids (Sias) are negatively charged functional monosaccharides present in a wide variety of natural sources (plants, animals and microorganisms). Sias play an important role in many life processes, which are widely applied in the medical and food industries as intestinal antibacterials, antivirals, anti-oxidative agents, food ingredients, and detoxification agents. Most Sias are composed of N -acetylneuraminic acid (Neu5Ac, >99%), and Sia is its most commonly used name. In this article, we review Sias in terms of their structures, applications, determination methods, metabolism, and production strategies. In particular, we summarise and compare different production strategies, including extraction from natural sources, chemical synthesis, polymer decomposition, enzymatic synthesis, whole-cell catalysis, and de novo biosynthesis via microorganism fermentation. We also discuss research on their physiological functions and applications, barriers to efficient production, and strategies for overcoming these challenges. We focus on efficient de novo biosynthesis strategies for Neu5Ac via microbial fermentation using novel synthetic biology tools and methods that may be applied in future. This work provides a comprehensive overview of recent advances on Sias, and addresses future challenges regarding their functions, applications, and production. [ABSTRACT FROM AUTHOR]

Abstract: A chemical investigation on the cephalaspidean Bulla occidentalis from the Gulf of Mexico led to known niuhinones A (1) and B (2) along with a new acyclic polypropionate, named niuhinone C (3). A complete NMR assignment of niuhinone A has been achieved by spectroscopic experiments, thus revising the Δ14 configuration. The occurrence of structurally related polypropionates in Bullidae species from different geographical areas is in agreement with a de novo origin in this family of molluscs and adds a further piece of evidence on prey–predator relationships among cephalaspideans. [Copyright &y& Elsevier]

Fatty acids are essential components of biological membranes, important for the maintenance of cellular structures, especially in organisms with complex life cycles like protozoan parasites. Apicomplexans are obligate parasites responsible for various deadly diseases of humans and livestock. We analyzed the fatty acids produced by the closest phototrophic relatives of parasitic apicomplexans, the chromerids Chromera velia and Vitrella brassicaformis, and investigated the genes coding for enzymes involved in fatty acids biosynthesis in chromerids, in comparison to their parasitic relatives. Based on evidence from genomic and metabolomic data, we propose a model of fatty acid synthesis in chromerids: the plastid-localized FAS-II pathway is responsible for the de novo synthesis of fatty acids reaching the maximum length of 18 carbon units. Short saturated fatty acids (C14:0–C18:0) originate from the plastid are then elongated and desaturated in the cytosol and the endoplasmic reticulum. We identified giant FAS I-like multi-modular enzymes in both chromerids, which seem to be involved in polyketide synthesis and fatty acid elongation. This full-scale description of the biosynthesis of fatty acids and their derivatives provides important insights into the reductive evolutionary transition of a phototropic algal ancestor to obligate parasites. [ABSTRACT FROM AUTHOR]

Indole-3-acetic acid (IAA) is a critical plant hormone that regulates cell division, development, and metabolism. IAA synthesis in plants and plant-associated microorganisms cannot fulfill the requirement for large-scale agricultural production. Here, two novel IAA biosynthesis pathways, tryptamine (TAM) and indole-3-acetamide (IAM), were developed for IAA production by whole-cell catalysis and de novo biosynthesis in an engineered Escherichia coli MG1655. When 10 g/L l-tryptophan was used as a substrate, an MIA-6 strain containing a heterologous IAM pathway had the highest IAA titer of 7.10 g/L (1.34 × 10 3 mg/g DCW), which was 98.4 times more than MTAI-5 containing the TAM pathway by whole-cell catalysis. De novo IAA biosynthesis was optimized by improving NAD(P)H availability, resulting in an increased IAA titer of 906 mg/L obtained by the MGΔadhE::icd strain, which is 29.7% higher than the control . These strategies exhibit the potential for IAA production in engineered E. coli and possible industrial applications.

Obligate intracellular pathogens have coevolved with their host, leading to clever strategies to access nutrients, to combat the host's immune response, and to establish a safe niche for intracellular replication. The host, on the other hand, has also developed ways to restrict the replication of invaders by limiting access to nutrients required for pathogen survival. In this review, we describe the recent advancements in both computational methods and high-throughput -omics techniques that have been used to study and interrogate metabolic functions in the context of intracellular parasitism. Specifically, we cover the current knowledge on the presence of amino acid biosynthesis and uptake within the Apicomplexa phylum, focusing on human-infecting pathogens: Toxoplasma gondii and Plasmodium falciparum . Given the complex multi-host lifecycle of these pathogens, we hypothesize that amino acids are made, rather than acquired, depending on the host niche. We summarize the stage specificities of enzymes revealed through transcriptomics data, the relevance of amino acids for parasite pathogenesis in vivo, and the role of their transporters. Targeting one or more of these pathways may lead to a deeper understanding of the specific contributions of biosynthesis versus acquisition of amino acids and to design better intervention strategies against the apicomplexan parasites.

Aldehydes are a class of highly versatile chemicals that can undergo a wide range of chemical reactions and are in high demand as starting materials for chemical manufacturing. Biologically, fatty aldehydes can be produced from fatty acyl-CoA by the action of fatty acyl-CoA reductases. The aldehydes produced can be further converted enzymatically to other valuable derivatives. Thus, metabolic engineering of microorganisms for biosynthesizing aldehydes and their derivatives could provide an economical and sustainable platform for key aldehyde precursor production and subsequent conversion to various value-added chemicals. Saccharomyces cerevisiae is an excellent host for this purpose because it is a robust organism that has been used extensively for industrial biochemical production. However, fatty acyl-CoA-dependent aldehyde-forming enzymes expressed in S. cerevisiae thus far have extremely low activities, hence limiting direct utilization of fatty acyl-CoA as substrate for aldehyde biosynthesis. Toward overcoming this challenge, we successfully engineered an alcohol-forming fatty acyl-CoA reductase for aldehyde production through rational design. We further improved aldehyde production through strain engineering by deleting competing pathways and increasing substrate availability. Subsequently, we demonstrated alkane and alkene production as one of the many possible applications of the aldehyde-producing strain. Overall, by protein engineering of a fatty acyl-CoA reductase to alter its activity and metabolic engineering of S. cerevisiae , we generated strains with the highest reported cytosolic aliphatic aldehyde and alkane/alkene production to date in S. cerevisiae from fatty acyl-CoA., (Copyright © 2020 Foo, Rasouliha, Susanto, Leong and Chang.)

Lipoic acid is a valuable organosulfur compound used as an antioxidant for dietary supplementation, and potentially anti-diabetic and anti-cancer. Currently, lipoic acid is obtained mainly through chemical synthesis, which requires toxic reagents and organic solvents, thus causing environmental issues. Moreover, chemically synthesized lipoic acid is conventionally a racemic mixture. To obtain enantiomerically pure R -lipoic acid, which has superior bioactivity than the S form, chiral resolution and asymmetric synthesis methods require additional reagents and solvents, and often lead to wastage of S -lipoic acid or precursors with undesired chirality. Toward sustainable production of R -lipoic acid, we aim to develop a synthetic biology-based method using engineered yeast. Here, we deepened mechanistic understanding of lipoic acid biosynthesis and protein lipoylation in the model yeast Saccharomyces cerevisiae to facilitate metabolic engineering of the microbe for producing free R -lipoic acid. In brief, we studied the biosynthesis and confirmed the availability of protein-bound lipoate in yeast cells through LC-MS/MS. We then characterized in vitro the activity of a lipoamidase from Enterococcus faecalis for releasing free R -lipoic acid from lipoate-modified yeast proteins. Overexpression of the lipoamidase in yeast mitochondria enabled de novo free R -lipoic acid production in vivo. By overexpressing pathway enzymes and regenerating the cofactor, the production titer was increased ∼2.9-fold. This study represents the first report of free R -lipoic acid biosynthesis in S. cerevisiae . We envision that these results could provide insights into lipoic acid biosynthesis in eukaryotic cells and drive development of sustainable R -lipoic acid production., (Copyright © 2020 Chen, Foo, Ling and Chang.)

Background: Pterostilbene, a structural analog of resveratrol, has higher oral bioavailability and bioactivity than that of the parent compound; but is far less abundant in natural sources. Thus, to efficiently obtain this bioactive resveratrol analog, it is necessary to develop new bioproduction systems., Results: We identified a resveratrol O-methyltransferase (ROMT) function from a multifunctional caffeic acid O-methyltransferase (COMT) originating from Arabidopsis, which catalyzes the transfer of a methyl group to resveratrol resulting in pterostilbene production. In addition, we constructed a biological platform to produce pterostilbene with this ROMT gene. Pterostilbene can be synthesized from intracellular L-tyrosine, which requires the activities of four enzymes: tyrosine ammonia lyase (TAL), p-coumarate:CoA ligase (CCL), stilbene synthase (STS) and resveratrol O-methyltransferase (ROMT). For the efficient production of pterostilbene in E. coli, we used an engineered E. coli strain to increase the intracellular pool of L-tyrosine, which is the initial precursor of pterostilbene. Next, we tried to produce pterostilbene in the engineered E. coli strain using L-methionine containing media, which is used to increase the intracellular pool of S-adenosyl-L-methionine (SAM). According to this result, pterostilbene production as high as 33.6 ± 4.1 mg/L was achieved, which was about 3.6-fold higher compared with that in the parental E. coli strain harboring a plasmid for pterostilbene biosynthesis., Conclusion: As a potential phytonutrient, pterostilbene was successfully produced in E. coli from a glucose medium using a single vector system, and its production titer was also significantly increased using a L-methionine containing medium in combination with a strain that had an engineered metabolic pathway for L-tyrosine. Additionally, we provide insights into the dual functions of COMT from A. thaliana which was characterized as a ROMT enzyme.

Biotic and abiotic stressors often result in the buildup of amino acid pools in plants, which serve as potential stress mitigators. However, the role of anabolic (de novo amino acid synthesis) versus catabolic (proteolytic) processes in contributing to free amino acid pools is less understood. Using stable isotope-resolved metabolomics (SIRM), we measured the de novo amino acid synthesis in glyphosate susceptible (S-) and resistant (R-) Amaranthus palmeri biotypes. In the S-biotype, glyphosate treatment at 0.4 kg ae/ha resulted in an increase in total amino acids, a proportional increase in both (14)N and (15)N amino acids, and a decrease in soluble proteins. This indicates a potential increase in de novo amino acid synthesis, coupled with a lower protein synthesis and a higher protein catabolism following glyphosate treatment in the S-biotype. Furthermore, the ratio of glutamine/glutamic acid (Gln/Glu) in the glyphosate-treated S- and R-biotypes indicated that the initial assimilation of inorganic nitrogen to organic forms is less affected by glyphosate. However, amino acid biosynthesis downstream of glutamine is disproportionately disrupted in the glyphosate treated S-biotype. It is thus concluded that the herbicide-induced amino acid abundance in the S-biotype is contributed by both protein catabolism and de novo synthesis of amino acids such as glutamine and asparagine.

Resveratrol is a well-known triphenolic natural product present in red wine. For its contribution to human health, the demand for resveratrol as a food and nutrition supplement has increased significantly. In recent years, the rapid development of synthetic biology has promoted extensive work to increase the production of resveratrol in microbes. However, supplementation of expensive phenylpropanoic precursors was required in current engineered strains. Here, we first utilized the site-specific integration strategy to produce resveratrol in Escherichia coli The genes tal, 4cl and sts were site-specific integrated into the loci of genes tyrR and trpED in the chromosome of E. coli BW25113 (DE3). The final strain was capable of producing 4.612 mg L(-1) of resveratrol from glucose., (© FEMS 2016. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

De novo biosynthesis of linoleic acid (LA) and its conversion to (Z,Z)-6,9-heptadecadiene were examined in Carpoglyphus lactis (Acarina, Carpoglyphidae). Experiments involving (13)C-administration using [1-(13)C]-d-glucose revealed that (13)C atoms were incorporated into LA of total lipid extracted from the mite, resulting in labeling of all even-numbered carbons. This result demonstrated that LA was produced from (13)C-labeled acetyl-CoA, which is indicative of direct de novo biosynthesis. In these feeding experiments involving [1-(13)C]-D-glucose, (13)C atoms were also incorporated into (Z,Z)-6,9-heptadecadiene, which is one of the major secretory components in the mite. The labeling pattern of (Z,Z)-6,9-heptadecadiene at odd-numbered carbons agreed well with that of LA after loss of the carboxyl carbon. It was concluded that the mites could stably convert LA into (Z,Z)-6,9-heptadecadiene without the dietary requirement of this essential fatty acid., (Copyright © 2013 Elsevier Ltd. All rights reserved.)