Biotechnology advances最新文献

Bacterial nanocellulose (BNC) is a biopolymer that is drawing significant attention for a wide range of applications thanks to its unique structure and excellent properties, such as high purity, mechanical strength, high water holding capacity and biocompatibility. Nevertheless, the biomanufacturing of BNC is hindered due to its low yield, the instability of microbial strains and cost limitations that prevent it from being mass-produced on a large scale. Various approaches have been developed to address these problems by genetically modifying strains and to produce BNC-based biomaterials with added value. These works are summarized and discussed in the present article, which include the overexpression and knockout of genes related and not related with the nanocellulose biosynthetic operon, the application of synthetic biology approaches and CRISPR/Cas techniques to modulate BNC biosynthesis. Further discussion is provided on functionalized BNC-based biomaterials with tailored properties that are incorporated in-vivo during its biosynthesis using genetically modified strains either in single or co-culture systems (in-vivo manufacturing). This novel strategy holds potential to open the road toward cost-effective production processes and to find novel applications in a variety of technology and industrial fields.

Astaxanthin is a valuable orange-red carotenoid with wide applications in agriculture, food, cosmetics, pharmaceuticals and nutraceuticals areas. At present, the biological synthesis of astaxanthin mainly relies on Haematococcus pluvialis and Xanthophyllomyces dendrorhous. With the rapid development of synthetic biology, more recombinant microbial hosts have been genetically constructed for astaxanthin production including Escherichia coli, Saccharomyces cerevisiae and Yarrowia lipolytica. As multiple genes (15) were involved in the astaxanthin synthesis, it is particularly important to adopt different strategies to balance the metabolic flow towards the astaxanthin synthesis. Furthermore, astaxanthin is a fat-soluble compound stored intracellularly, hence efficient extraction methods are also essential for the economical production of astaxanthin. Several efficient and green extraction methods of astaxanthin have been reported in recent years, including the superfluid extraction, ionic liquid extraction and microwave-assisted extraction. Accordingly, this review will comprehensively introduce the advances on the astaxanthin production and extraction by using different microbial hosts and strategies to improve the astaxanthin synthesis and extraction efficiency.

A plethora of CRISPR effectors, such as Cas3, Cas9, and Cas12a, are commonly employed as gene editing tools. Among these, Cas12 effectors developed based on Class II type V proteins exhibit distinct characteristics compared to Class II type VI and type II effectors, such as their ability to generate non-allelic DNA double-strand breaks, their compact structures, and the presence of a single RuvC-like nuclease domain. Capitalizing on these advantages, Cas12 family proteins have been increasingly explored and utilized in recent years. However, the characteristics and applications of different subfamilies within the type V protein family have not been systematically summarized. In this review, we focus on the characteristics of type V effector (CRISPR/Cas12) proteins and the current methods used to discover new effector proteins. We also summarize recent modifications based on engineering of type V effectors. In addition, we introduce the applications of type V effectors for gene editing in animals and plants, including the development of base editors, tools for regulating gene expression, methods for gene targeting, and biosensors. We emphasize the prospects for development and application of CRISPR/Cas12 effectors with the goal of better utilizing toolkits based on this protein family for crop improvement and enhanced agricultural production.

Microalgae are a group of microorganisms, mostly photoautotrophs with high CO₂ fixation capacity, that have gained increased attention in the last decades due to their ability to produce a wide range of valuable metabolites, such as carotenoids and polyunsaturated fatty acids, for application in food/feed, pharmaceutical, and cosmeceutical industries. Their increasing relevance has highlighted the importance of identifying and culturing new bioactive-rich microalgae species, as well as of a thorough understanding of the growth conditions to optimize the biomass production and master the biochemical composition according to the desired application. Thus, this review intends to describe the main cell processes behind the production of carotenoids and polyunsaturated fatty acids, in order to understand the possible main triggers responsible for the accumulation of those biocompounds. Their economic value and the biological relevance for human consumption are also summarized. In addition, an extensive review of the impact of culture conditions on microalgae growth performance and their biochemical composition is presented, focusing mainly on the studies involving Pavlovophyceae species. A complementary description of the biochemical composition of these microalgae is also presented, highlighting their potential applications as a promising bioresource of compounds for large-scale production and human and animal consumption.

Adenosine triphosphate (ATP) regeneration is a significant step in both living cells and in vitro biotransformation (ivBT). Rotary motor ATP synthases (ATPases), which regenerate ATP in living cells, have been widely assembled in biomimetic structures for in vitro ATP synthesis. In this review, we present a comprehensive overview of ATPases, including the working principle, orientation and distribution density properties of ATPases, as well as the assembly strategies and applications of ATPase-based ATP regeneration modules. The original sources of ATPases for in vitro ATP regeneration include chromatophores, chloroplasts, mitochondria, and inverted Escherichia coli (E. coli) vesicles, which are readily accessible but unstable. Although significant advances have been made in the assembly methods for ATPase-artificial membranes in recent decades, it remains challenging to replicate the high density and orientation of ATPases observed in vivo using in vitro assembly methods. The use of bioproton pumps or chemicals for constructing proton motive forces (PMF) enables the versatility and potential of ATPase-based ATP regeneration modules. Additionally, overall robustness can be achieved via membrane component selection, such as polymers offering great mechanical stability, or by constructing a solid supporting matrix through layer-by-layer assembly techniques. Finally, the prospects of ATPase-based ATP regeneration modules can be expected with the technological development of ATPases and artificial membranes.

Increased consumer awareness for healthier and more sustainable products has driven the search for naturally sourced compounds as substitutes for chemically synthesized counterparts. Research on pigments of natural origin, such as carotenoids, particularly lutein, has been increasing for over three decades. Lutein is recognized for its antioxidant and photoprotective activity. Its ability to cross the blood-brain barrier allows it to act at the eye and brain level and has been linked to benefits for vision, cognitive function and other conditions. While marigold flower is positioned as the only crop from which lutein is extracted from and commercialized, microalgae are proposed as an alternative with several advantages over this terrestrial crop. The main barrier to scaling up lutein production from microalgae to the commercial level is the low productivity compared to the high costs. This review explores strategies to enhance lutein production in microalgae by emphasizing the overall productivity over lutein content alone. Evaluation of how culture parameters, such as light quality, nitrogen sufficiency, temperature and even stress factors, affect lutein content and biomass development in batch phototrophic cultures was performed. Overall, the total lutein production remains low under this metabolic regime due to the low biomass productivity of photosynthetic batch cultures. For this reason, we describe findings on microalgal cultures grown under different metabolic regimes and culture protocols (fed-batch, pulse-feed, semi-batch, semi-continuous, continuous). After a careful literature examination, two-step heterotrophic or mixotrophic cultivation strategies are suggested to surpass the lutein productivity achieved in single-step photosynthetic cultures. Furthermore, this review highlights the urgent need to develop technical feasibility studies at a pilot scale for these cultivation strategies, which will strengthen the necessary techno-economic analyses to drive their commercial production.

The bioprocessing industry is undergoing a significant transformation in its approach to quality assurance, shifting from the traditional Quality by Testing (QbT) to Quality by Design (QbD). QbD, a systematic approach to quality in process development, integrates quality into process design and control, guided by regulatory frameworks. This paradigm shift enables increased operational efficiencies, reduced market time, and ensures product consistency. The implementation of QbD is framed around key elements such as defining the Quality Target Product Profile (QTPPs), identifying Critical Quality Attributes (CQAs), developing Design Spaces (DS), establishing Control Strategies (CS), and maintaining continual improvement. The present critical analysis delves into the intricacies of each element, emphasizing their role in ensuring consistent product quality and regulatory compliance.

The integration of Industry 4.0 and 5.0 technologies, including Artificial Intelligence (AI), Machine Learning (ML), Internet of Things (IoT), and Digital Twins (DTs), is significantly transforming the bioprocessing industry. These innovations enable real-time data analysis, predictive modelling, and process optimization, which are crucial elements in QbD implementation. Among these, the concept of DTs is notable for its ability to facilitate bi-directional data communication and enable real-time adjustments and therefore optimize processes. DTs, however, face implementation challenges such as system integration, data security, and hardware-software compatibility. These challenges are being addressed through advancements in AI, Virtual Reality/ Augmented Reality (VR/AR), and improved communication technologies.

Central to the functioning of DTs is the development and application of various models of differing types – mechanistic, empirical, and hybrid. These models serve as the intellectual backbone of DTs, providing a framework for interpreting and predicting the behaviour of their physical counterparts. The choice and development of these models are vital for the accuracy and efficacy of DTs, enabling them to mirror and predict the real-time dynamics of bioprocessing systems. Complementing these models, advancements in data collection technologies, such as free-floating wireless sensors and spectroscopic sensors, enhance the monitoring and control capabilities of DTs, providing a more comprehensive and nuanced understanding of the bioprocessing environment.

This review offers a critical analysis of the prevailing trends in model-based bioprocessing development within the sector.

Carbon source is crucial for the cell growth and metabolism in microorganisms, and its utilization significantly affects the synthesis efficiency of target products in microbial cell factories. Compared with a single carbon source, co-utilizing carbon sources provide an alternative approach to optimize the utilization of different carbon sources for efficient biosynthesis of many chemicals with higher titer/yield/productivity. However, the efficiency of bioproduction is significantly limited by the sequential utilization of a preferred carbon source and secondary carbon sources, attributed to carbon catabolite repression (CCR). This review aimed to introduce the mechanisms of CCR and further focus on the summary of the strategies for co-utilization of carbon sources, including alleviation of CCR, engineering of the transport and metabolism of secondary carbon sources, compulsive co-utilization in single culture, co-utilization of carbon sources via co-culture, and evolutionary approaches. The findings of representative studies with a significant improvement in the bioproduction of chemicals via the co-utilization of carbon sources were discussed in this review. It suggested that by combining rational metabolic engineering and irrational evolutionary approaches, co-utilizing carbon sources can significantly contribute to the bioproduction of chemicals.

Biological production of hydrogen has a tremendous potential as an environmentally sustainable technology to generate a clean fuel. Among the different available methods to produce biohydrogen, dark fermentation features the highest productivity and can be used as a means to dispose of organic waste biomass. Within this approach, Clostridia have the highest theoretical H₂ production yield. Nonetheless, most strains show actual yields far lower than the theoretical maximum: improving their efficiency becomes necessary for achieving cost-effective fermentation processes. This review aims at providing a survey of the metabolic network involved in H₂ generation in Clostridia and strategies used to improve it through metabolic engineering. Together with current achievements, a number of future perspectives to implement these results will be illustrated.

Enzymes play a pivotal role in various industries by enabling efficient, eco-friendly, and sustainable chemical processes. However, the low turnover rates and poor substrate selectivity of enzymes limit their large-scale applications. Rational computational enzyme design, facilitated by computational algorithms, offers a more targeted and less labor-intensive approach. There has been notable advancement in employing rational computational protein engineering strategies to overcome these issues, it has not been comprehensively reviewed so far. This article reviews recent developments in rational computational enzyme design, categorizing them into three types: structure-based, sequence-based, and data-driven machine learning computational design. Case studies are presented to demonstrate successful enhancements in catalytic activity, stability, and substrate selectivity. Lastly, the article provides a thorough analysis of these approaches, highlights existing challenges and potential solutions, and offers insights into future development directions.