Biochemistry & Physiology: Open Access
Open Access

Metabolic engineering; Enzyme cascade; Cascade biocatalysis; Multienzyme

Introduction

Engineering living organisms to produce useful chemicals, from high-value natural products like cannabinoids to low-value products like fuels, plastics, and building block chemicals, has received significant attention The drawbacks of extracting natural resources from native sources, such as agricultural boom and bust cycles, as well as the need for resource-intensive and pricey purification, would be eliminated by microbial production of natural products. Additionally, microbial production may be better for the environment than chemical synthesis. The replacement of high-volume petroleum products like fuels and commodity chemicals by metabolically engineered microbes will likely have the greatest impact on the environment [1-3]. There are various likely natural advantages: The starting materials for biomass can be used again; reduced emissions of greenhouse gases can be achieved by substituting non-petroleum-based starting materials; and the majority of the products are biodegradable. However, because cost is so important, replacing low-value, high-volume products is also the most expensive. To make bioderived items financially aggressive will require high-effectiveness transformation of the info biomass into valuable mixtures.

However, it is challenging for biological organisms to achieve efficient biomass conversion. There are numerous regulatory mechanisms in the cell that control pathway flow rates, making it difficult to optimize volumetric productivity due to the complexity of cellular physiology (see Glossary). The distribution of resources away from the desired products by life processes reduces yield. In addition, product titer can be restricted by intermediate or product toxicities [4]. Despite the usefulness of engineering regulatory mechanisms and competing pathways, the problem’s complexity is difficult to overcome. Even successful efforts to engineer strains that are industrially scalable typically required years of work from large teams and hundreds of individuals. Although living things clearly have the ability to integrate complex inputs and carry out remarkably complex tasks, it may make more sense for engineering purposes to draw inspiration from biology rather than try to harness life itself. For instance, we are able to observe how birds fly and learn from them, but airplane flight mechanisms do not imitate those of birds, so the outcome is better suited to our requirements and is easier to engineer. Similar to the cellfree, synthetic biochemistry approach, this one aims to create simpler, more streamlined versions of biologically inspired systems that can be efficiently engineered for use in everyday life. Cell-free methods still face many obstacles, but research into their potential has only begun. Here, we talk about how bio manufacturing of high-volume chemicals could benefit from cell-free production.

The cascade enzymes are expressed, purified, and then mixed in the purified activity approach. The enzymes can be expressed simultaneously or separately using the lysate method and the cells containing the desired enzymes are then lysed to release their contents. After that, the cascade enzyme-rich crude lysate can be utilized without purification. In most cases, undesirable enzyme activities must be eliminated in some way to prevent their conversion into undesirable side effects. There are options for hybrid approaches. A lysate containing other enzymes, for instance, might benefit from the addition of purified enzymes. Another strategy is to use a lysate for the majority of the enzymes and then add other enzymes that are expressed separately to the lysate. Naturally, this method is the most adaptable and could be especially useful in situations where some enzymes can only be expressed in a different organism while the majority of enzymes express in one organism [5-8]. High yields, titers, and productivityimportant production parameters that in part define commercial viability—may be more easily achieved by cell-free systems. Ideally, all of the input biomass (such as sugar, for example) would be converted into the desired product in order to achieve high yields. Because there is only one pathway in the bioreactor in the cell-free method, yields can be nearly 100 percent, with enzyme specificity and thermodynamics acting as the only limitations. Numerous biochemical pathways in cells divert sugar into undesirable side products that are necessary for life processes. In fact, deleting these side pathways is a common part of metabolic engineering, which can be challenging if you want to keep cells alive. Dynamic metabolic control, an alternative strategy, aims to separate the growth and production phases so that undesirable cellular pathways can be shut down during production.

In a cell-free system, identifying issues and addressing them is typically simpler. Because enzymes can be directly assayed, changes in their activities can be easily assessed. When compared to cellular systems, the mixture of metabolites is greatly simplified, making it simple to measure concentrations. Cofactor pools, such as redox states, can also be monitored. Any identified bottlenecks can frequently be resolved quickly by replacing a regulated enzyme with an unregulated one or a more stable enzyme with one that rapidly loses activity [9]. On the off chance that compound designing is required, it tends to be clear to present the recently rebuilt catalyst and optimize the framework. In a cell-free system, the mixture of metabolites is simpler than in a fermentation broth, making downstream processing simpler— especially for products that would normally be retained in cells. In addition, the use of organic overlays can be more adaptable because there is no need to worry about killing cells and less risk associated with emulsions. In point of fact, product isolation can be as simple as extracting practically pure product from an organic overlay.

Before synthetic biochemistry systems involving more than a few enzymes are ready for large-scale commercial manufacturing, extensive technical developments will be required (see Outstanding Questions). Most of the time, the problems of stabilizing enzymes and making enzymes on a large scale can probably be solved, but some classes of enzymes will need more fundamental work before they can be used in bio manufacturing. There has been little prior experience scaling complex enzyme systems to the levels required for commodity chemical production [10]. As we attempt to scale more complex systems, there is no doubt that there will be a lot to learn. Is it preferable to work at a low temperature, where microbial growth mitigation becomes necessary, or at a high temperature, despite the resulting cofactor instability? Is it possible to effectively scale aerobic systems that require oxygen dispersion? When is it appropriate to affix enzymes to a sturdy support? The most pressing requirement for cell-free bio manufacturing is probably the advancement of cofactor technology.

Conflict of Interest

The authors declared that there is no conflict of interest

Acknowledgment

None

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