Project Design

Golden gate collection that enables rapid production validation across strains

Synthetic biology to enable low carbon and efficient production of natural products is receiving increasing attention from relevant sectors. By introducing exogenous metabolic pathways, we enable the production of target products by the chosen chassis organisms. Currently, the commonly used chassis organisms include E. coli, Streptomyces, Vibrio natriegens, Bacillus subtilis, Saccharomyces cerevisiae, and Picornicus.

Since different chassis organisms have different metabolic networks and primary metabolic levels, they may exhibit several-fold different yields for the same exogenous metabolic pathway, so they need to be screened to obtain the optimal solution. In addition, the promoter sequences between prokaryotes and eukaryotes have large differences, which also limit the screening of chassis. Further, in industry, methods to integrate gene clusters into the genome are commonly used to reduce the antibiotics required for plasmid maintenance or to avoid metabolic deficiencies due to nutritional defects. For Saccharomyces cerevisiae, this can be done by homologous recombination, while for prokaryotes, additional enzymes such as the Cas12k transposase system are required for gene recombination, which in turn is dependent on the amount of enzyme expression and in the vast majority of cases requires additional testing as well as validation screening steps to get the right strain. In summary, the factors discussed above limit the rapid parallel testing of chassis organisms and become a limiting factor for efficient strain screening, testing, and development.

Golden gate, as a mature and convenient way to assemble molecular clones, can achieve large-scale metabolic pathway assembly in one go. Meanwhile, splice transfer is a prevalent method among bacteria that enables plasmid delivery. For gene cluster integration, transposases such as phiC31 are often used for the rapid integration of exogenous large fragment gene clusters in Streptomyces. Combining both of these with the Golden gate allows for parallel testing of different prokaryotic chassis organisms. By using artificially designed hybrid promoters, the same shuttle plasmid can be expressed in both prokaryotic organisms and yeast. If this type of promoter and the components required for shuttle plasmids can be introduced into the Golden gate system mentioned above, rapid parallel testing between prokaryotic chassis organisms and yeast can be achieved.

Here, we hope to build the ONCE collection, a fast and efficient Golden gate system for multi-chassis testing. Our Golden gate system has the following features:

1. contains special promoters and terminators;
2. contains elements required for splice transfer and elements required for shuttle plasmids;
3. loads systems that allow for rapid exchange between plasmid backbones and genomic integration vectors.

To be compatible with the currently known Golden gate collection, we designed our system based on the Marburg collection.

The Marburg Collection, proposed by the 2018 Marburg team, has well-established prokaryotic expression elements. In order to expand its cross-species functionality, we made the following modifications:

1. Introduce hybrid promoters compatible with prokaryotes and brewer's yeast to the Promoter module;
2. Add short synthetic terminators for yeast to the terminator module;
3. Add elements that can be used for splice transfer and replication in yeast to the ORI module;
4. Add recombinase recognition module and yeast homologous recombination module to the connector module;
5. Add nutritional defect screening tool to the antibiotic resistance module;
6. Introduce inducible promoters and corresponding regulators.

ORI compatible with conjugation and shuttling into yeast

When selecting yeast-prokaryotic hybrid promoters, and for single cis-trans yeast expression, we considered the following points:

1. first, these eukaryotic components should not interfere with bacterial expression;
2. second, sequence length was minimized to reduce synthesis costs;
3. third, the negative impact of untranslated sequences on bacterial mRNA stability was minimized;
4. fourth, we prevented homologous recombination by minimizing sequence homology to bring about fragment deletions by minimizing sequence homology;
5. fifth, potential termination signals, promoters, and ribosome binding sites were predicted in each part to avoid them as much as possible.

ORI compatible with splice transfer, yeast shuttle

We first consider splice transfer ORIs. currently, the only reported ORIs that can pass to bacteria by splice transfer are pBBR1 and R6K, of which PBBR1 has a higher broad spectrum but a single selectable copy number.

For E. coli, there are many ORIs with different copy numbers that can work, and it would provide a better tool for the study of non-model microorganisms if they could be changed to ORIs that can pass to non-model microorganisms by splice transfer. OriT is the element in the splice transfer plasmid that governs the occurrence of splice transfer, and it does this by triggering the rolling loop replication. That is, the combination of OriT with other ORIs could enable ORIs with other copy numbers to shuttle between E. coli as well as non-model bacteria. If the backbone is further supplemented with CEN/PK or 2u ORIs capable of replication in yeast, shuttling in yeast can be achieved.

Efficient strategies for rapid genome integration

CRISPR/Cas9 is often used to knock in segments to the genome in bacteria, and Cas12k, discovered in recent years, has also been developed for knocking in large segments. However, both technologies rely on the expression of Cas-related proteins and may not be applicable to some ribosome-depleted strains.

Tn7 is an enzyme capable of random insertion of fragments within the genome, and although it can insert sequences rapidly on the genome and proceed to the next step, it may disrupt some critical genes; also, the expression of Tn7 is highly toxic and may lead to many adaptive mutations, which may affect the work of metabolic pathways. CRISPR-CAST can be used for multicopy insertion of multiple loci where the number of inserted copies is positively correlated with culture time.

Tyrosine recombinases, such as Cre and Flp, are widely used for targeted genomic manipulation but need to be engineered to overcome their inherent response bidirectionality, which can lead to the re-excision of the integration product. In contrast, Large serine recombinases, such as Bxb1 and PhiC31, catalyze the unidirectional integration of DNA into matching sites. while PhiC31 can integrate its payload into pseudosite sites in the eukaryotic genome similar to its native attachment site, Bxb1 requires pre-installation of its preferred attachment site in the human genome18.

In the mean time, these events can also be achieved on bacterial genomes. The most classical transposase from E. coli, TnsABCD, is able to integrate fragments to attTn7, while most prokaryotes have attB sites that are recognized by PhiC31 and undergo integration of fragments.

By combining the properties of CRISPR-CAST and recombinases, we designed the strategy shown in the following figure.

Recombinant enzyme recognition module and yeast homologous recombination module

In fact, recomRecombinant enzyme recognition module and yeast homologous recombination modulebinases can perform not only single-site but also multi-site recombination, taking PhiC31 as an example.

Whereas the integration sites in Saccharomyces cerevisiae have been better elucidated, from which we can select suitable loci for.

Yeast screening module

For yeast, common screening markers are URA3, HIS3, Leu2, Trp1, KanMX. By deleting them upstream of the promoter, their expression in yeast can be reduced, which in turn boosts the copy number of the plasmid. By different combinations, different plasmid expressions can be achieved and the functions of the whole metabolic system can be regulated.

P4, TP3, TP2, and TP1 corresponded to the use of 20, 30, 50,and 100 bp sequences upstream of the start codon as the partiallydefective promoters, respectively)

Inducible promoter and the corresponding regulator

Because of human practice, we learned that many metabolic pathways are manipulated using inducible promoters. After research, we selected the Marionette collection and included them in our ONCE collection.

The Marionette collection consists of 12 optimized regulators and corresponding proteins with good coordination, lower leaky expression and higher response levels.

A general strategy for the construction of high FPP-producing and downstream terpenic chassis strains

Terpenoids have important biological functions and applications. Modern medical research has found that many terpenes have a wide range of anticancer effects. In addition to its antibacterial and anti-inflammatory activities, cucurbitacin B has been shown to inhibit the growth of human malignant tumor cells, including breast cancer cells, head and neck squamous carcinoma, pancreatic cancer, liver cancer, osteosarcoma and myeloid leukemia. To date, several cucurbitacins and cucurbitacin derivatives of Cucurbitaceae origin have been isolated.

Terpenoids also have a wide range of pharmacological effects such as anti-inflammatory and hypoglycemic. Triterpene saponins have been found to be anti-inflammatory, anti-allergic, leukemic, anti-viral, hypoglycemic, and against cardiovascular diseases. In addition, many plant-derived terpenoids are aromatic volatile substances, so they are widely used in fragrances, perfumes, flavorings and cosmetics, etc. Nootkatone, sclareol, menthol, linalool and other aromatic terpenes are important components of plant essential oils, and are also the source of odor of fruit aroma and plant flower fragrance, so they are aromatic foods and they are commonly used as fragrances in aromatic foods and essential oil cosmetics.

The structural diversity of terpenoids also makes them advanced substitutes for fuels such as gasoline and diesel. In fact, farnes-ene, bisabolene, pinene, isopentenol, and isopentanol are all recognized as precursors for fuels and fuels.

In recent years, the synthesis of terpenoids has become one of the hot topics in the field of biology. Here, we propose a trinity FPP high yield strategy combining endogenous FPP synthesis pathway optimization, exogenous synthesis pathway introduction and artificial synthesis pathway integration.

We have designed a strategy that can substantially enhance FPP production. In nature, the MVA pathway as well as the MEP pathway are common FPP supply pathways. In bacteria, the MEP pathway is the common FPP synthesis pathway, while in yeast, it is basically dominated by MVA. For bacteria, we can introduce the MVA pathway in it to initially enhance FPP supply; for yeast strains, over-expression of MVA upstream genes is often the main focus.

On the other hand, the path starting from glucose to DMAPP and IPP is not the easiest in terms of chemical modifications. In fact, not from a biological point of view, but from a synthetic chemistry point of view, it does not make sense to start from glucose to reach DMAPP or IPP. At this point, the simplest way to obtain minimal structural modifications of DMAPP and IPP is to use dimethylallyl alcohol (dMAOH) and isopentenol (IOH), the two homologous alcohols of DMAPP and IPP, and to perform a double phosphorylation. The paradigm of terpenoid bioproduction may change if a one- or two-step enzymatic phosphorylation starts from DMAOH and IOH into DMAPP and IPP. The first enzyme using phosphatase as an artificial pathway to obtain DMAPP/IPP from DMAOH/IOH in combination with IPK. In one example, the gene encoding the PhoN phosphatase from Shigella and IPK from Thermoplasma acidophilum was cloned into the pETDuet plasmid (forming the so-called alcohol-dependent hemiterpene (ADHP) pathway). This plasmid was then combined with a plasmid carrying a gene encoding the lycopene bio-synthetic pathway and allowing a lycopene titer of 150 mg/L at the end of fermentation in E. coli to optimize the concentration of added alcohols (DMAOH and IOH, 5 mM each) to further increase the lycopene titer by 190 mg/L.

When it comes to the synthesis of downstream products, due to their strong hydrophobicity, terpenoids are often incompatible with the aqueous phase environment of the cytoplasm when they are synthesized in microorganisms and accumulate in large quantities, which can lead to a decrease in terpenoid yields. For example, the lycopene synthesis pathway was introduced in yeast in addition to overexpression of triglyceride synthase. The yield of lycopene was greatly enhanced, demonstrating that increasing the intracellular hydrophobic environment enhances the biosynthesis of hydrophobic products. Using the property of β-caveolin-1 protein (β-Cav1) to form caveolar membranes with phospholipid molecules intracellularly, the multienzyme caveolar membranes were formed by attaching/assembling Idi, IspA, and AFS with an action peptide added to one end of β-Cav1, thus creating a hydrophobic microenvironment for these enzymes. The modified enzymes were shown to create a hydrophobic microenvironment. It was demonstrated that the modified strain achieved an approximately 10-fold increase in farnesene production.

In summary, we plan to improve the synthesis of FPP precursors as well as the production of downstream products by exogenously expressing the MVA pathway, incorporating an artificially designed IUP pathway, and through a modular strategy of increasing the intracellular hydrophobic environment.

FPP Fluorescent Reporting System

FPP is the widest synthesized substrate for terpenoids. However, the concentration of FPP is highly dynamic in cells, and indirect quantification of FPP using FPP downstream products is limited by the system's complexity, making it difficult to obtain convincing results. This made it difficult for us to develop a high-throughput screen for high FPP-producing strains, so we created a split FPP-sensing complex referencing the design of Anum et al.[1] and replaced the mouse dihydrofolate reductase in the article with a couple of split β-barrel fluorescent proteins for a more intuitive coupling of FPP to the fluorescent signal. This reporter effectively enhanced the throughput of our high FPP-producing strain construction and can be used for subsequent screening of other more efficient terpene synthases.

Vibrio natriegens and seawater culture

This experiment has been submitted to check-in form and approved.

Vibrio natriegens is a non-pathogenic marine microorganism with about half the replication time of E. coli and is able to accumulate more biomass, as well as being less susceptible to phage infestation . The above characteristics make it a great potential for metabolic engineering, industrial fermentation, capable of generating large economic benefits, and has received more and more attention in the past year.

Vibrio natriegens requires high environmental salinity (salt ions of about 2-4%) and has a good tolerance to low-intensity UV radiation, while being compatible with a wide range of temperature conditions (25°C-37°C) and also has nitrogen fixation. Currently, the most commonly used strain is ATCC 14048, and a total of four transformation methods have been developed: electro-transformation, chemical transformation, splice transfer, and natural transformation . In addition, the nucleic acid endonuclease expressed in the dns gene in Vibrio natriegens significantly affects the transformation efficiency, and knocking out the dns can increase the transformation efficiency of the plasmid by up to 10,000-fold. Another factor limiting the efficient use of Vibrio natriegens is the uncertainty of whether some antibiotics will work or not, and the corresponding working concentration. Condex DNA performed a large-scale genomic fragment knockdown of wild-type Vibrio natriegens using natural transformation as well as recombinant enzymes, resulting in strain Vmax X2, which is characterized by a defective dns gene, high efficiency of exogenous protein expression, and stable culture screening conditions.

In terms of genetic elements, several papers have characterized the workings of promoters, ribosome binding sites, terminators, and replication initiation sites in Vibrio natriegens, adequately meeting the need for synthetic biology modification . Meanwhile, the TCA cycle in biochemical metabolism as well as partial sugar metabolism has been elucidated. Many authors have used Vibrio natriegens as a chassis organism for the microbial ab initior synthesis of natural or industrial products. However, the rapid growth state allows its endogenous metabolic pathways to exhibit large variation and may contain metabolic pathways that have not been identified in other organisms.

In addition, due to the high growth rate of Vibrio natriegens, it needs to continuously synthesize and degrade endogenous proteins, which means that the endogenous protein degradation mechanism needs to work continuously, thus the exogenous protein introduced through genetic engineering can easily compete with the native protein degradation mechanism and affect the metabolism of the bacterium to a large extent, and the exogenous protein work is also affected.

The current metabolic engineering production method relies on autoclave of culture media and fermentation equipment, and has very strict requirements for operators and fermentation procedures. Non-sterile open fermentation does not require sterilization of fermentation equipment and media, and is considered by some scholars to be a promising next-generation industrial biotechnology with practical applications due to its advantages of simple fermentation process and energy cost savings.

Seawater from different regions may have different trace elements that act on the growth of Vibrio natriegens. In addition, some seafood farms also discharge high-salt farm wastewater, which is also suitable for the growth as well as metabolism of Vibrio natriegens.

Here we tested the growth of Vibrio natriegens in different regions of seawater and farm drainage, and will soon proceed with the production of lycopene and alpha-Bisabolol.