Enhanced production of ectoine from methane using metabolically engineered Methylomicrobium alcaliphilum 20Z

Background Ectoine (1,3,4,5-tetrahydro-2-methyl-4-pyrimidinecarboxylic acid) is an attractive compatible solute because of its wide industrial applications. Previous studies on the microbial production of ectoine have focused on sugar fermentation. Alternatively, methane can be used as an inexpensive and abundant resource for ectoine production by using the halophilic methanotroph, Methylomicrobium alcaliphilum 20Z. However, there are some limitations, including the low production of ectoine from methane and the limited tools for the genetic manipulation of methanotrophs to facilitate their use as industrial strains. Results We constructed M. alcaliphilum 20ZDP with a high conjugation efficiency and stability of the episomal plasmid by the removal of its native plasmid. To improve the ectoine production in M. alcaliphilum 20Z from methane, the ectD (encoding ectoine hydroxylase) and ectR (transcription repressor of the ectABC-ask operon) were deleted to reduce the formation of by-products (such as hydroxyectoine) and induce ectoine production. When the double mutant was batch cultured with methane, ectoine production was enhanced 1.6-fold compared to that obtained with M. alcaliphilum 20ZDP (45.58 mg/L vs. 27.26 mg/L) without growth inhibition. Notably, a maximum titer of 142.32 mg/L was reached by the use of an optimized medium for ectoine production containing 6% NaCl and 0.05 μM of tungsten without hydroxyectoine production. This result demonstrates the highest ectoine production from methane to date. Conclusions Ectoine production was significantly enhanced by the disruption of the ectD and ectR genes in M. alcaliphilum 20Z under optimized conditions favoring ectoine accumulation. We demonstrated effective genetic engineering in a methanotrophic bacterium, with enhanced production of ectoine from methane as the sole carbon source. This study suggests a potentially transformational path to commercial sugar-based ectoine production. Graphical Abstract

Seed-Borne Cercospora beticola can Initiate Cercospora Leaf Spot from Sugar Beet (Beta vulgaris L.) Fruit Tissue

Cercospora leaf spot (CLS) is a globally important disease of sugar beet (Beta vulgaris L.) caused by the fungus Cercospora beticola. Long-distance movement of C. beticola has been indirectly evidenced in recent population genetic studies, suggesting potential dispersal via seed. Commercial sugar beet “seed” consists of the reproductive fruit (true seed surrounded by maternal pericarp tissue) coated in artificial pellet material. In this study, we confirmed the presence of viable C. beticola in sugar beet fruit for 10 of 37 tested seed lots. All isolates harbored the G143A mutation associated with quinone outside inhibitor resistance and 32 of 38 isolates had reduced demethylation inhibitor sensitivity (EC50 > 1 µg/ml). Planting of commercial sugar beet seed demonstrated the ability of seed-borne inoculum to initiate CLS in sugar beet. Cercospora beticola DNA was detected in DNA isolated from xylem sap, suggesting the vascular system is used to systemically colonize the host. We established nuclear ribosomal internal transcribed spacer region amplicon sequencing using the MinION platform to detect fungi in sugar beet fruit. Fungi from 19 different genera were identified from 11 different sugar beet seed lots, but Fusarium, Alternaria, and Cercospora were consistently the three most dominant taxa, comprising an average of 93% relative read abundance over 11 seed lots. We also present evidence that C. beticola resides in the pericarp of sugar beet fruit, rather than the true seed. The presence of seed-borne inoculum should be considered when implementing integrated disease management strategies for CLS of sugar beet in the future.

Enhanced Production of Ectoine From Methane Using Metabolically Engineered Methylomicrobium Alcaliphilum 20Z

Background: Ectoine (1,3,4,5-tetrahydro-2-methyl-4-pyrimidinecarboxylic acid) is an attractive compatible solute because of its wide industrial applications. Previous studies on the microbial production of ectoine have focused on sugar fermentation. Alternatively, methane can be used as an inexpensive and abundant resource for ectoine production by using the halophilic methanotroph, Methylomicrobium alcaliphilum 20Z. However, there are some limitations, including the low production of ectoine from methane and the limited tools for the genetic manipulation of methanotrophs to facilitate their use as industrial strains.Results: We constructed a genetically tractable M. alcaliphilum 20Z with a high conjugation efficiency and stability of the episomal plasmid by the removal of its native plasmid. To improve the ectoine production in M. alcaliphilum 20Z from methane, the ectD (encoding ectoine hydroxylase) and ectR (transcription repressor of the ectABC-ask operon) were deleted to reduce the formation of by-products (such as hydroxyectoine) and induce ectoine production. When the double mutant was batch cultured with methane, ectoine production was enhanced 1.6-fold compared to that obtained with M. alcaliphilum 20ZDP (45.58 mg/L vs. 27.26 mg/L) without growth inhibition. Notably, the use of an optimized medium for ectoine production, containing 6% NaCl and 0.05 µM tungsten, gave ectoine yields of up to 142.32 mg/L without hydroxyectoine production. This result demonstrates the highest ectoine production from methane to date.Conclusions: Ectoine production was significantly enhanced by the disruption of the ectD and ectR genes in M. alcaliphilum 20Z under optimized conditions favoring ectoine accumulation. We demonstrated effective genetic engineering in a methanotrophic bacterium, with enhanced production of ectoine from methane as the sole carbon source. This study suggests a potentially transformational path to commercial sugar-based ectoine production.

Lipozyme 435-Mediated Synthesis of Xylose Oleate in Methyl Ethyl Ketone

In this paper, we have performed the Lipozyme 435-catalyzed synthesis of xylose oleate in methyl ethyl ketone (MEK) from xylose and oleic acid. The effects of substrates’ molar ratios, reaction temperature, reaction time on esterification rates, and Lipozyme 435 reuse were studied. Results showed that an excess of oleic acid (xylose: oleic acid molar ratio of 1:5) significantly favored the reaction, yielding 98% of xylose conversion and 31% oleic acid conversion after 24 h-reaction (mainly to xylose mono- and dioleate, as confirmed by mass spectrometry). The highest Lipozyme 435 activities occurred between 55 and 70 °C. The predicted Ping Pong Bi Bi kinetic model fitted very well to the experimental data and there was no evidence of inhibitions in the range assessed. The reaction product was purified and presented an emulsion capacity close to that of a commercial sugar ester detergent. Finally, the repeated use of Lipozyme 435 showed a reduction in the reaction yields (by 48 and 19% in the xylose and oleic acid conversions, respectively), after ten 12 h-cycles.

To bee or not to beet with neonicotinoids

In January 2021 the UK government granted an application for authorisation to use thiamethoxam, a neonicotinoid pesticide, to protect commercial sugar beet crops from attack by viruses transmitted by aphids. This was the first time such an authorisation had been granted in the United Kingdom (UK) and there were concerns that it signalled a weakening of environmental standards now that the UK was no longer part of the European Union. In fact, similar authorisations had been granted by several European Member States in the last 2 years, despite the ban on the use of neonicotinoids introduced in 2018. Nevertheless, the reasons for granting the authorisation do suggest that the balance between adopting a precautionary approach to environmental protection and taking emergency action to protect economic interests may have shifted. It was acknowledged that the proposed mitigation to safeguard bees and other wildlife was not entirely satisfactory. In the end, due to unforeseen weather conditions it meant that the pesticide is not necessary, which in itself demonstrates that short-term emergency measures are unsuitable for dealing with the problem. If the sugar beet industry is to continue to prosper in the UK, it will need to be managed in a way that provides resistance to virus infection without the use of controversial chemicals.

The Impact of Sugar Particles Size and Natural Substitutes for the Replacement of Sucrose and Fat in Chocolate Brownies: Sensory and Physicochemical Analysis

As fat contributes important textural properties such as lubricity and tenderness to cakes, it is plausible to focus on ways to increase the perception of these properties with the aim of creating the illusion of a higher fat. The utilisation of small sugar particles has been shown to increase the moist and soft texture of Chocolate Brownies. The present study assessed three different sugar particle sizes in their ability to create the illusion of fat content and therefore their ability to permit fat replacement (FR) in this product. The unground commercial sugar (200-5181 µm) was used as the control (UC) and two of its sieved sugar separates, Large (L924-1877 µm) and Small (S459-972 µm) were investigated. For each, fat was replaced using pureed black beans. The most accepted sample was used for sucrose replacement (SR) using inulin and Rebaudioside A. (Reb A.). Samples containing the smallest sugar fraction with 25% FR were most significantly associated overall acceptability (OA) (p<0.01). The application of small sugar particles did not significantly negatively affect OA or liking of samples at a level of 75% FR compared to the other two sugar fractions. The utilisation of small sugar particles (459-972 µm) in the preparation of baked goods could aid baking & industry professionals in reducing the fat content of cake-like products.

Chapter 8 - Sweet commercial drink adoption by urban Chinese middle-class people – Between social control and new beverage consumption contexts

This chapter focuses on the consumption practices of Chinese urban middle-class people regarding sweet commercial drinks. A qualitative study conducted between 2014 and 2016 revealed that bland boiled water is considered relatively healthy but is not evocative of conviviality, in contrast to commercial beverages and sugar which are considered more unhealthy, but associated with pleasure and conviviality. This ambivalence appears in the representations of sweet commercial drinks, especially around the symbol of balance between traditional Chinese medicine and dietetics and industrial modernity. This chapter addresses the new social and family tensions that have emerged regarding beverage and sugar management. It highlights the settings in which commercial sugar-containing beverages have been embedded in the consumption patterns of middle-class city dwellers in China.

Quality and Nutritional Value of Functional Strawberry Marmalade Enriched with Chia Seed (Salvia hispanica L.)

The aim of the study was to develop and characterize marmalade having functional food ingredients such as omega-3 fatty acid, dietary fiber, protein, and antioxidants with the addition of chia seed (Salvia hispanica L.). During the development of marmalade formulations, sweetener type and chia content in the strawberry marmalade were decided by two-step sensory analysis. In the first step, four different formulas were prepared separately by using sorbitol, isomalt, commercial Stevia™ powder, and isomalt together with sorbitol. The control formula was prepared with sucrose (refined commercial sugar). In the first part of the study, sensorial parameters showed good acceptability for sorbitol. Thereafter, in the second step, marmalades were prepared with 2.5% and 5% (by weight) chia seed including sorbitol. According to sensory panels, sorbitol had the highest acceptance level and the chia seed content was chosen to be used as 5% in the formulations. Chia seed and sorbitol addition increased the phenolic content by 15.45% and the dietary fiber content by 168% and decreased the caloric value by 48% compared to the control prepared with sucrose and without chia seed. The final product had 1.5% omega-3 fatty acid and could be declared as “omega-3 source” in the label. The viscosity of chia-added marmalade was found to be slightly higher than the viscosity of control, even though there was no distinct difference between the two samples. The gel-like character was more dominant in chia-added strawberry marmalade compared to the control. The addition of chia at 5% may contribute to the crosslinking without formation of a gel structure.

Mealybug species (Hemiptera: Coccomorpha: Pseudococcidae) on soursop and sugar apple (Annonaceae) in North-East Brazil, with description of a new species of Pseudococcus Westwood

Several species of Annonaceae are economically important fruit-tree crops in North-East Brazil, including Pernambuco state. However, in several regions within the state, the fruits are commonly infested by mealybugs (Hemiptera: Coccomorpha: Pseudococcidae). There is a lack of information about the mealybug species damaging this produce, so a survey of mealybug species associated with commercial sugar apple (Annona squamosa L.) and soursop (A. muricata L.) was conducted in the main production areas. The species Ferrisia virgata (Cockerell), Maconellicoccus hirsutus (Green), Planococcus minor (Maskell), Phenacoccus solenopsis Tinsley and Pseudococcus jackbeardsleyi Gimpel & Miller were found on both Annona species. Dysmicoccus brevipes (Cockerell), Ferrisia dayslirii Kaydan & Gullan and Ferrisia malvastra (MacDaniel) were found only on soursop; and Ferrisia cristinae Kaydan & Gullan, Planococcus citri (Risso), Pseudococcus annonae sp. n. Pacheco da Silva & Kaydan and Pseudococcus sp. were found only on sugar apple. The species F. cristinae, F. dasylirii, F. malvastra and Ph. solenopsis are recorded infesting these hosts for the first time. The most abundant mealybug species found were F. virgata, Pl. minor, Ps. jackbeardsleyi and M. hirsutus, often forming heavy infestations and damaging the fruits. A new species, Pseudococcus annonae sp. n. Pacheco da Silva & Kaydan, is described and illustrated, and an identification key to the mealybug genera occurring on annonaceous species in the Neotropical region is also provided.