生态工程中食物链组合的环分析

本文研究了一个用于治理水体生态系统富营养化的生态工程,应用环分析方法对工程中的食物链的组合进行了分析。在这里,贝类被用于清除过量的藻类;种植水生植物改变营养的流转途径并且放养食植性鱼类,通过捕捞鱼和贝,使水体中的有机物和无机盐沿着从藻类到贝类和从水生植物到食植性鱼类两条途径由水体生态系统中输出,应用环分析的手段,可以调整生态工程中食物链的组合结构,使整个生态工程具有某种特定的功能。     

Metabolic engineering of Mannheimia succiniciproducens for malic acid production using dimethylsulfoxide as an electron acceptor

Microbial production of various TCA intermediates and related chemicals through the reductive TCA cycle has been of great interest. However, rumen bacteria that naturally possess strong reductive TCA cycle have been rarely studied to produce these chemicals, except for succinic acid, due to their dependence on fumarate reduction to transport electrons for ATP synthesis. In this study, malic acid (MA), a dicarboxylic acid of industrial importance, was selected as a target chemical for mass production using Mannheimia succiniciproducens, a rumen bacterium possessing a strong reductive branch of the TCA cycle. The metabolic pathway was reconstructed by eliminating fumarase to prevent MA conversion to fumarate. The respiration system of M. succiniciproducens was reconstructed by introducing the Actinobacillus succinogenes dimethylsulfoxide (DMSO) reductase to improve cell growth using DMSO as an electron acceptor. Also, the cell membrane was engineered by employing Pseudomonas aeruginosa cis-trans isomerase to enhance MA tolerance. High inoculum fed-batch fermentation of the final engineered strain produced 61 g/L of MA with an overall productivity of 2.27 g/L/h, which is the highest MA productivity reported to date. The systems metabolic engineering strategies reported in this study will be useful for developing anaerobic bioprocesses for the production of various industrially important chemicals.     

A well-characterized polycistronic-like gene expression system in yeast

Efficient expression of multiple genes is critical to yeast metabolic engineering for the bioproduction of bulk and fine chemicals. A yeast polycistronic expression system is of particular interest because one promoter can drive the expression of multiple genes. 2A viral peptides enable the cotranslation of multiple proteins from a single mRNA by ribosomal skipping. However, the wide adaptation of 2A viral peptides for polycistronic-like gene expression in yeast awaits in-depth characterizations. Additionally, a one-step assembly of such a polycistronic-like system is highly desirable. To this end, we have developed a modular cloning (MoClo) compatible 2A peptide-based polycistronic-like system capable of expressing multiple genes from a single promoter in yeast. Characterizing the bi-, tri-, and quad-cistronic expression of fluorescent proteins showed high cleavage efficiencies of three 2A peptides: E2A from equine rhinitis B virus, P2A from porcine teschovirus-1, and O2A from Operophtera brumata cypovirus-18. Applying the polycistronic-like system to produce geraniol, a valuable industrial compound, resulted in comparable or higher titers than using conventional monocistronic constructs. In summary, this highly-characterized polycistronic-like gene expression system is another tool to facilitate multigene expression for metabolic engineering in yeast.     

Dynamic genome-scale modeling of Saccharomyces cerevisiae unravels mechanisms for ester formation during alcoholic fermentation

Fermentation employing Saccharomyces cerevisiae has produced alcoholic beverages and bread for millennia. More recently, S. cerevisiae has been used to manufacture specific metabolites for the food, pharmaceutical, and cosmetic industries. Among the most important of these metabolites are compounds associated with desirable aromas and flavors, including higher alcohols and esters. Although the physiology of yeast has been well-studied, its metabolic modulation leading to aroma production in relevant industrial scenarios such as winemaking is still unclear. Here we ask what are the underlying metabolic mechanisms that explain the conserved and varying behavior of different yeasts regarding aroma formation under enological conditions? We employed dynamic flux balance analysis (dFBA) to answer this key question using the latest genome-scale metabolic model (GEM) of S. cerevisiae. The model revealed several conserved mechanisms among wine yeasts, for example, acetate ester formation is dependent on intracellular metabolic acetyl-CoA/CoA levels, and the formation of ethyl esters facilitates the removal of toxic fatty acids from cells using CoA. Species-specific mechanisms were also found, such as a preference for the shikimate pathway leading to more 2-phenylethanol production in the Opale strain as well as strain behavior varying notably during the carbohydrate accumulation phase and carbohydrate accumulation inducing redox restrictions during a later cell growth phase for strain Uvaferm. In conclusion, our new metabolic model of yeast under enological conditions revealed key metabolic mechanisms in wine yeasts, which will aid future research strategies to optimize their behavior in industrial settings.     

Thermostability enhancement of polyethylene terephthalate degrading PETase using self- and nonself-ligating protein scaffolding approaches

Polyethylene terephthalate (PET) hydrolase enzymes show promise for enzymatic PET degradation and green recycling of single-use PET vessels representing a major source of global pollution. Their full potential can be unlocked with enzyme engineering to render activities on recalcitrant PET substrates commensurate with cost-effective recycling at scale. Thermostability is a highly desirable property in industrial enzymes, often imparting increased robustness and significantly reducing quantities required. To date, most engineered PET hydrolases show improved thermostability over their parental enzymes. Here, we report engineered thermostable variants of Ideonella sakaiensis PET hydrolase enzyme (IsPETase) developed using two scaffolding strategies. The first employed SpyCatcher-SpyTag technology to covalently cyclize IsPETase, resulting in increased thermostability that was concomitant with reduced turnover of PET substrates compared to native IsPETase. The second approach using a GFP-nanobody fusion protein (vGFP) as a scaffold yielded a construct with a melting temperature of 80°C. This was further increased to 85°C when a thermostable PETase variant (FAST PETase) was scaffolded into vGFP, the highest reported so far for an engineered PET hydrolase derived from IsPETase. Thermostability enhancement using the vGFP scaffold did not compromise activity on PET compared to IsPETase. These contrasting results highlight potential topological and dynamic constraints imposed by scaffold choice as determinants of enzyme activity.     

Effects of temperature on metabolic rate and lower dissolved oxygen tolerance of juvenile speckled peacock bass Cichla temensis

The speckled peacock bass Cichla temensis is a popular sport and food fish that generates substantial angling tourism and utilitarian harvest within its range. Its popularity and value make this species important for management and a potential aquaculture candidate for both fisheries enhancement and food fish production. However, little is known of optimal physiochemical conditions in natural habitats, which also are important for the development of hatchery protocols for handling, spawning and grow-out. Speckled peacock bass have been documented to have high sensitivity to extreme temperatures, but the metabolic underpinnings have not been evaluated. In this study, the effects of temperature (25, 30 and 35°C) on the standard metabolic rate (SMR) and lower dissolved oxygen tolerance (LDOT) of juvenile speckled peacock bass (mean ± standard error total length 153 ± 2 mm and wet weight 39.09 ± 1.37 g) were evaluated using intermittent respirometers after an acclimation period of 2 weeks. Speckled peacock bass had the highest SMR at 35°C (345.56 ± 19.89 mgO₂ kg⁻¹ h⁻¹), followed by 30°C (208.16 ± 12.45 mgO₂ kg⁻¹ h⁻¹) and 25°C (144.09 ± 10.43 mgO₂ kg⁻¹ h⁻¹). Correspondingly, the Q₁₀, or rate of increase in aerobic metabolic rate (MO₂) relative to 10°C, for 30–35°C was also greater (2.76) than from 25 to 30°C (2.08). Similarly, speckled peacock bass were the most sensitive to hypoxia at the warmest temperature, with an LDOT at pO₂ of 90 mmHg (4.13 mg l⁻¹) at 35°C compared to pO₂ values of 45 mmHg (2.22 mg l⁻¹) and 30 mmHg (1.61 mg l⁻¹) at 30 and 25°C, respectively. These results indicate that speckled peacock bass are sensitive to temperatures near 35°C, therefore we recommend managing and rearing this species at 25–30°C.