With the deepening of the concept of "green development", "green biological manufacturing" has gradually become the main direction of the transformation of the next generation of manufacturing.
Synthetic biology technology has led to a more efficient biomanufacturing process, manufacturing products including amino acids, antibiotics, polymeric materials, renewable chemicals, high-value natural products, pharmaceutical intermediates, etc., covering almost every aspect of daily life.
By mining genetic elements, optimizing genetic circuits, and modifying chassis cells, synthetic biologists can synthesize engineered microbes "from the bottom up" and function orientation, and continuously engineer them for leaner fermentation pathways and more productive "cell factories."
"I have been engaged in the research of microbial breeding and fermentation before, and the synthetic products are naturally synthesized by microorganisms, and the blindness of using traditional breeding techniques is too great to select high-yielding strains. To this end, I wondered whether I could use modern genetic engineering technology to synthesize microorganisms into products that could not be synthesized before. And then optimize the pathway to increase its yield? Professor Liu Jianzhong, director of the Institute of Synthetic Biology at the School of Life Sciences of Sun Yat-sen University and deputy director of the Biomedical Center of Sun Yat-sen University, said in an interview with Shenghui SynBio.
Figure 丨 Liu Jianzhong (Source: Interviewee)
Liu Jianzhong is one of the earliest scholars in China to start studying synthetic biology, as early as 2006, his team tried to introduce exogenous gene clusters into E. coli, and produced coenzyme Q10, lycopene, β-carotene, zeaxanthin, astaxanthin and other substances.
At present, Liu Jianzhong's main research direction is high-value natural products, which mainly include two categories: one is aromatic compounds with strong antioxidant, bacteriostatic and other activities, and the other is carotenoids.
<h1 class="pgc-h-arrow-right" data-track="15" > synthetic biology intelligent breeding</h1>
Before the development and maturity of synthetic biology breeding technology, traditional breeding technology seems to have a large "luck" component, whether it is natural selection or mutagenic breeding, the resulting strains are likely to evolve or degenerate, and the efficiency is low; while hybrid breeding can accumulate dominant mutations, but its operation is complex, so it has not been applied on a large scale.
The problems mentioned by Professor Liu Jianzhong above are now one of the application ideas of synthetic biology. On the basis of mastering the genome information of chassis cells, the corresponding gene clusters are adjusted, unlike single-point gene editing, the gene editing technology of synthetic biology is often to increase, add, delete, subtract, or even introduce foreign gene clusters to achieve the adjustment of certain functions of chassis cells or give them functions that they do not have.
(Source: cell)
Compared with traditional microbial breeding methods, synthetic biology brings more intelligent breeding.
Up to now, Liu Jianzhong's team has used the combination of metabolic engineering, synthetic biology and systems biology to build a number of international leading technology level terpene-producing and aromatic compound microbial cell factories.
Cell factories are the final form of synthetic biology fermentation technology, the main body of which is the engineered microorganisms that have been modified. Industrialized cell factories emphasize metrics such as conversion rate, production rate, and yield, so optimization research around chassis cells is also the focus of the industry.
<h1 class="pgc-h-arrow-right" data-track="73" > improves chassis cell energy utilization</h1>
The transformation of chassis cells involves many aspects, such as reducing the expression of non-essential genes, reducing intracellular endogenous consumption, reducing the toxicity of products to cells, and so on.
In April, Liu's team published an article in Biotechnology for Biofuels that the yield of the target product was increased by designing and modifying chassis cells to improve cellular energy utilization, that is, the utilization of ATP and NADPH during biosynthesis.
Intracellular biosynthesis reactions are inseparable from the participation of coenzyme factors such as ATP and NADPH, and in the case of sufficient substrates, ATP will become a speed-limiting step for biological reactions.
The target product of this study is 4-hydroxyphenylacetic acid (4HPAA), a valuable natural aromatic compound. In chassis cells, synthesis of 1 mol 4HPAA requires the involvement of 2 mol ATP and 1 mol NADPH, which are designed to reduce the consumption of ATP and NADPH in other biological reactions, focusing on the main reactions that produce 4HPAA.
To this end, crispR interference technology was used to silence 80 NADPH consumption and 400 ATP consumption genes, and to screen for genes that do not affect cell survival and can increase 4HPAA production. Subsequent knockout of these genes can further increase yields, but the deletion of certain essential genes can lead to cellular malnutrition.
Therefore, the study introduced a quorum sensing system, so that the expression of essential genes gradually decreased with the increase of the number of cell populations, and finally after this series of modifications, the yield of 4HPAA could reach up to 28.57g/L, and the yield was 27.64% (mol/mol), which was higher than the highest value reported in the literature.
The article names this series of methods CECRiS (cofactor engineering based on CRISPRi screening).
"This method can also be used for other high value-added product synthesis, because most of the biosynthesis process requires the consumption of ATP and NADPH, requiring the participation of enzymes, so it can be optimized through such a process, and the method is more versatile." Liu Jianzhong added.
Liu Jianzhong also revealed that in his latest study, the use of a cell-free system to synthesize 4HPAA can double the yield.
He explained, "Cell-free synthetic biology is a synthetic biology technology developed in recent years, for high toxicity, heterogeneous pathway mismatch, mass transfer restrictions, by-product competition pathways and other issues of the product, the use of living cell yield is low, can not achieve industrialization, the use of cell-free synthesis can avoid the above defects of live cell synthesis, may be a better synthetic pathway." ”
<h1 class="pgc-h-arrow-right" data-track="74" > need policies to promote industrialization</h1>
Last year, Liu Jianzhong became the person in charge of the National Key Research and Development Program synthetic biology key project "Design Principles and Applications of Robust Artificial Gene Components" Project 4 (Integration and Application of Robust Artificial Gene Components).
Liu Jianzhong shared that one of the purposes of synthetic biology is to standardize and modularize biological components like radio, so that anyone can more easily access and apply them. This key project is to establish a robust microbial cell factory, so that the synthetic biological components and devices carried by the cells can play a stable role in the large-scale industrial production process.
Figure丨 Cell Factory (Source: Nature)
Liu Jianzhong believes that under the current demand for "green biomanufacturing", the biomanufacturing route of bulk APIs and bulk chemicals is an application field that is relatively easy to achieve transformation and landing, so some provincial-level projects are mainly designed around these two industries.
"With the funding of national key research and development and government research projects at all levels, some scholars have built microbial cell factories that can be industrialized, and my research results are currently cooperating with enterprises to carry out industrialization research and development."
At the same time, he also pointed out that "domestic synthetic biology has achieved remarkable results, especially in the aspect of "creation to know", some of which have been in the international leader. However, there is still a big gap between the 'application of creation', especially the industrialization, and the developed countries in Europe and the United States. ”
"In terms of food and food additives, there are no relevant regulations in China to obtain production licenses, which limits its industrialization process. Now more and more microbial strains are constructed using synthetic biology, and only by solving these problems can the technology be applied to actual production as soon as possible. ”
"The good news is that people in the field are also aware of this problem and are actively promoting it, but it will take some time."