Design

Background

    In order to combat climate change,all mankind has formulated a series of conventions and clauses to deal with this problem. China aims at achieve a carbon peak by 2030 and achieve carbon neutrality by 2060. The massive use of fossil energy is one of the main culprits of carbon emissions, and the use of renewable substances to produce biofuels to gradually replace fossil energy is an effectve means to reduce carbon emissions. We set oursights on the aviation industry.

    Sustainable aviation fuel (SAF) refers to the fossil fuel substitute generated by chemical reaction of various sustainable and repeatedly obtained raw materials. CO2 is recycled from the raw material to realize the carbon cycle, which is regarded as one of the most promising measures to reduce the carbon emissions of the aviation industry quickly.

    The traditional method of producing isobutanol mainly relies on fossil energy such as oil and natural gas as raw materials, and produces a lot of carbon dioxide in the process. This project uses microorganisms to synthesize isobutanol, with mild reaction conditions and renewable raw materials, which is conducive to the development of isobutanol biofuel industry, reducing carbon emissions, and thus contributing to "carbon peak and carbon neutrality".

    In conclusion,we have developed a zero-carbon cellulose aviation fuel production model.With Z.mobilis as the chassis organism,while producing isobutanol,it fixed the CO2 in the atmosphere to reduce carbon emissions.

Overview

    We designed two systems during the project. At first,We replaced the promoter of A4 plasmid to achieve update iteration, and at the same time added one copy of isobutanol production gene kdcA in the genome to achieve double copy to further improve isobutanol production.Then to construct the carbon sequestration section, we designed a carbon dioxide fixation pathway within the strain. We selected the most common and deeply studied Calvin cycle pathway, and to improve carbon fixation efficiency, we decided to construct carboxysome in the strain for CO2 concentration. Therefore, our construction is completed from two aspects.

Figure 1 A metabolic pathway constructed in Zymomonas mobilis

    In the metabolic pathway diagram, red arrows indicate enhanced isobutanol synthesis, gray arrows indicate inhibition of ethanol production, and blue indicates enhanced carbon fixation.An explanation of the illustrated enzyme is shown below.

Table 1 Enzyme interpretation

Gene	Enzyme
Als	acetolactate synthase
IlvC	ketol-acid reductoisomerase
IlvD	dihydroxy-acid dehydratase
KdcA	2-ketoacid decarboxylase
Pdc	pyruvate dehydrogenase
Adh	alcohol dehydrogenase
Adhs	alcohol dehydrogenases
XylA	xylose isomerase
XylB	xylanase
Rpe	phosphoribulose epimerase
PrkA	phosphoribulokinase
Rubisco	1,5-2 phosphate ribulose carboxylase
CA	carbonic anhydrase

Isobutanol Biosynthesis - Zymomonas mobilis

Design Overview

    By introducing key enzyme genes of isobutanol metabolism pathway into ZM4 strain to construct the isobutanol production pathway, we designed two parts simultaneously: transforming plasmids carrying related enzymes genes, replacing corresponding enzyme genes at genomic sites in ZM4 strain.Finally we introduced CRISPR-ddCpf1 to inhibit the ethanol metabolism pathway of ZM4 strain, achieving the transfer of carbon flow from ethanol production to isobutanol production.

Why produce isobutanol?

    Sustainable aviation fuel (SAF) is the fundamental approach and the most important measure for the civil aviation industry to cope with climate change and achieve carbon emission reduction. Among various biofuels, isobutanol has the advantages of high heat density, high combustion value, low volatility and low moisture absorption compared with ethanol, and is regarded as a higher performance biofuel that can replace ethanol.

Selection of chassis organism

    Zymomonas mobilis was originally isolated from pulque, Mexico. It is the only known Gram-negative bacteria that can metabolize ethanol by Entner-Doudoroff (ED) pathway under anaerobic conditions.The ED pathway has fewer thermodynamic constraints than the EMP pathway and requires fewer enzyme proteins to maintain the same flow. It has the characteristics of high ethanol fermentation rate, high apparent sugar yield, great ethanol tolerance and biosafety (GRAS).

    In recent years, Z.mobilis is being developed as a cell factory for bioenergy and various bio-based chemicals, and has received widespread attention. The high ethanol production performance of Z.mobilis made the strain naturally have a very high tolerance to alcohols. The unique physiological characteristics and excellent production characteristics of Z.mobilis demonstrate its potential as a high-yielding cell factory for isobutanol, so our project uses it as our chassis organism.

Table 2 Chassis species comparison table

Parameters	Z. mobilis	E.coli	S. cerevisiae
Growth Condition	Facultative anaerobe	Facultative aerobic Movable	Facultative aerobic Movable
Safe States	safety	unsafety	safety
Ethanol Productivity（/g/h）	5.67	0.60	0.67
Ethanol Ratio	98%	88%	90–93%
Ethanol Tolerance（v/v）	16%	6%	15%

Plasmid optimization

    Building on the foundations of previous iGEM teams and laboratories, we started with the A4 plasmid(pEZ-A4-Ptet-Bsals-RBS-ilvC-RBS-ilvD) for optimization. Literature results show that heterogene Bsals is the key gene to increase isobutanol production through overexpression of isobutanol metabolism pathway related genes, but endogenous genes ilvC and ilvD are also indispensable.

    The Z.mobilis reducing isomerase gene ilvC, dihydroxylate dehydratase gene ilvD, and acetolactate synthase gene Bsals from Bacillus subtilis were constructed into the A4 plasmid. The genes were all expressed by inducible promoters and can be transferred to ZM4 strain to express isobutanol related genes.

    Due to the low expression yield ( PART-IMPROVEMENT for details), we decide to optimize and modify the plasmid

Promoter optimization

    To avoid the use of tetracycline to achieve the isobutanol production, the constitutive strong promoter of Peno will be added, Construction of A5 plasmid(pEZ15A-Ptet-Bsals2-Peno-ilvC-ilvD).Then, our strategy was replacing the Ptet promoter on the A5 plasmid with constitutive promoter Ppdc and PBAD and constructing the A6 plasmid (pEZ15A-Ppdc-Bsals2-Peno-ilvC-ilvD) and A7 plasmid(pEZ15A-PBAD-Bsals2-Peno-ilvC-ilvD).

    We found the Ppdc and PBAD combination strong promoter and predicted that it would greatly help improve isobutanol production.

CRISPRi system

    Considering that the ethanol metabolism pathway can divert carbon flux, another strategy is needed to divert carbon flux from ethanol production to isobutanol biosynthesis. The pdc gene is a key gene in the ethanol metabolism pathway, so we decided to use the CRISPRi system. We use dcpf1 protein and gRNA to silence the pdc gene to increase the isobutanol production.
Method Steps：
①　Substituting dCpf1 related genes with strain ZMO0038 site, constructing strain 0038:: Ptet dcpf1.
②　Transforming plasmid pEZ39P-cpf1-gRNA pdc carrying gRNA into the strain

Dual copy kdca replacement

    We obtained the exogenous keto decarboxylase kdcA gene from Lactococcus lactis, which is a key enzyme in the isobutanol production pathway and can induce and catalyze the final steps of isobutanol formation, To enhance its expressive effect， we decided to increase the gene copy number in the genome ZMO1650，ZMO1547 site.

CO2 Concentrating and Fixation

Design Overview

    Considering the problem of carbon dioxide emissions in the production process of isobutanol, the project also considers introducing the Calvin cycle pathway and Carboxysome to concentrate carbon and fix carbon dioxide in the production process of isobutanol, so that more carbon flow to the production of isobutanol and realize the efficient production of zero-carbon isobutanol.

    We constructed the Calvin-Benson-Bassham cycle(CBB cycle) in Z.mobilis and introduced carboxylator genes from cyanobacteria at same time to improve carbon sequestration efficiency.

Selection of chassis organism

    Zymomonas mobilis8b is an ethanol pathogenic bacterium that can simultaneously utilize glucose and xylose. It was modified from the engineered strain Zymomonas mobilis to utilize xylose and fully utilize resources. In order to enable Z.mobilis to utilize xylose, an engineered Z.mobils8b was constructed, expressing heterologous genes of talB, tktA, and xylAB from Escherichia coli for xylose utilization. And ZM8b strain can construct Calvin Benson Basham on the basis of xylose utilization pathway

    In the literature, it was found that the growth time of Z.mobilis8b was much longer than that of ZM4 original bacteria, and more carbon sources were needed for metabolic growth. Therefore, the Calvin cycle pathway was introduced to fix the carbon dioxide in the isobutanol production process, allowing more carbon sources to flow towards the production of isobutanol, achieving efficient production of zero carbon isobutanol.So we constructed the ZM8b strain as a chassis cell.

Construction of Calvin Benson Bassham (CCB) pathway

Key enzyme—Rubisco

    Ribulose-1,5-bisphosphocarboxylase/oxygenase (Rubisco) is a rate limiting enzyme in the Calvin cycle pathway, playing an important role in the Calvin cycle and determining the rate of carbon assimilation in plant photosynthesis. This enzyme synthesizes 3-phosphoglycerate by catalyzing CO2 consumption of ATP and reduced coenzyme II.

    By searching for Rubisco related gene sequences in the database, the gene rbc of ribose-1,5-diphosphate carboxylase/oxygenase (Rubisco) was found_Tb, we constructed the gene on the pEZ39p Core plasmid and transformed it into Z. mobilis 8b.

Phosphoribosylkinase gene prkA and CA gene

    On this basis, we introduce the phosphoribokinase gene prkA to construct a complete Calvin cycle carbon sequestration pathway. Phosphate ribokinase can catalyze ribose-5-phosphate to Ribulose-1,5-2P, and then consume CO2 to complete the reactionunder the catalysis of ribose-1,5-diphosphate carboxylase. We introduce the carbonic anhydrase gene CA, which can convert intracellular HCO3- to CO2.

Carboxysome—CO2 concentration mechanism

    We have reviewed relevant literature and learned that some organisms undergoing the Calvin cycle have CO2 concentration mechanisms. Due to Rubisco being an inefficient enzyme with slow catalytic speed and poor ability to distinguish between competing substrates CO2 and O2, many Calvin cycle organisms require other auxiliary links to help improve carbon dioxide fixation efficiency. And our Z.mobilis8b also draws on learning and constructs its own CO2 concentration mechanism – carboxysome.

Figure.2 Coupling 5-carbon glucose metabolism and Calvin cycle

Construction of carboxysome shell protein

    Through consulting materials, we learned about the carboxysome of blue-green algae (Synechococus elongatus PCC 7942) and chose to introduce carboxysome genes from blue-green algae in order to improve carbon sequestration efficiency In NCBI （ https://www.ncbi.nlm.nih.gov/ ） In the database search for carboxyl matrix related gene sequences, carboxyl matrix protein related genes ccmK1, ccmK2, ccmO, ccmL, ccmM, and ccmN were obtained from Halotec sp. PCC 7418, and the carbonic anhydrase gene sequence was synthesized from Synechococus elongatus PCC 7942.

Figure.3 Carboxylator carbon concentration mechanism

Rubisco Connection of various components

— inducing shell protein encapsulation to wrap Rubisco

    ccmM is a linker protein that binds Rubisco to the shell protein by interacting with the recruitment protein ccmN, inducing the shell protein to encapsulate Rubisco. We constructed the carboxylate linker gene ccmM, recruitment protein gene ccmN, and the gene rbc for ribose-1,5-diphosphate carboxylase/oxygenase (Rubisco) in plasmid 39p-core_TB is controlled by the inducible promoter Ptet and expressed in the presence of the inducer tetracycline.

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