Science: Chinese scientists have made starch from carbon dioxide!

A team led by Yanhe Ma of Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences has designed and constructed an 11-step unnatural carbon sequestration and starch synthesis pathway from scratch in a similar way of "building blocks", and achieved the complete synthesis from carbon dioxide to starch molecule in the laboratory for the first time. NMR and other tests showed that the structure of synthetic starch was consistent with that of natural starch. Preliminary laboratory tests show that synthetic starch is about 8.5 times more efficient than starch produced by conventional agriculture. Under the condition of sufficient energy supply, according to the current technical parameters, the annual production of starch of 1 cubic meter bioreactor is equivalent to the annual production of starch of 5 mu of corn land in China. This new route makes it possible to shift starch production from traditional agricultural cultivation to industrial manufacturing, opening up a new technical route for synthesizing complex molecules from CO2.

The research results, entitled "cell-free chemostarch Synthesis from Carbon Dioxide", were published in the latest issue of Science and reported by Xinhua News and Science and Technology Daily.

[Scheme design]

Research team used a similar "building blocks" approach, using chemical catalysts will high concentration of carbon dioxide in high density under the action of hydrogen reduction of carbon compounds (C1), and then build a new polymerization enzyme carbon by design, according to the chemical reaction principle exhibited a carbon compounds aggregated into carbon 3 (C3), the final optimization through biological way. The tritecarboxylic compounds are then polymerized into carbon six (C6) compounds, which are further synthesized into amylose and amylopectin (Cn) compounds.

The team used formalase (FLS) to design and construct the enzymatic portion of the starch synthesis pathway from candidate C1 intermediates and drafted two concise starch synthesis pathways from formic acid or methanol using combinational algorithms. In principle, starch can be synthesized by a nine-core reaction of CO2 with formic acid or methanol as a bridging intermediate for C1 (Figure 1, inner circle). Specifically, the C1 modules (for formaldehyde production), C3 modules (for D-glyceraldehyde 3-phosphate production), C6 modules (for D-glucose 6-phosphate production) and Cn modules (for starch synthesis). However, through retrieval and simulation, the authors found that the formaldehyde produced by the energy-saving but thermodynamically unfavorable C1 module may not provide the material for the critical reaction of FLS in the C3a module. Therefore, they constructed an alternative C1 module with a thermodynamically more favorable reaction cascade. The thermodynamically most favorable C1e module was successfully assembled with the C3a module and a significantly higher yield of C3 compounds was obtained from methanol. With the help of computational pathway design, the team established the artificial Starch anabolic Pathway (ASAP) 1.0, with 10 methanol-based enzymatic reactions, by assembling and replacing 11 modules consisting of 62 enzymes from 31 organisms (Figure 1, outer circle). The main intermediates and target products of ASAP1.0 were detected by isotope 13C labeling, which verified the full function of starch synthesis from methanol.

                                                                Figure 1. Design and modular assembly of synthetic starch anabolic pathways

Solving bottleneck problems, ASAP 1.0 Advanced ASAP 2.0

After establishing ASAP 1.0, the research team attempted to optimize this approach by addressing potential bottlenecks. First, due to its low kinetic activity, the enzyme FLS accounts for approximately 86% of the total protein dose in ASAP 1.0 to maintain metabolic flux and keep toxic formaldehyde at very low levels. Directional evolution increased the catalytic activity of FLS, resulting in a variant flS-M3, whose activity increased by 4.7 times. Figure 2B-D shows that the variant FBP-AR contains two mutations at the AMP allosteric site, which can reduce ADP inhibition and significantly improve G-6-P production of DHA. The inhibition patterns of FBP and FBP-AR by three nucleotides showed that ATP or ADP were the decisive factors of systemic inhibition. By integrating FBP-AR with a reported variant resistant to G-6-P, the combined variant FBP-AGR was further improved. Considering ATP competition between DAK and ADP-glucose pyrophosphorylase (AGP), starch production was abnormally reduced during the first 4 hours due to an increase in substrate DHA and its kinase DAK (Figure 2A). We demonstrate that the coexistence of DHA and DAK severely inhibits starch synthesis through Cnb (Figure 2E) and exports DHA phosphate (DHAP) as the main starch product (Figure 2F, column 1), which confirms that DAK competively consumes most ATP. Instead of reducing the use of DAK, the authors tried to enhance the ability of AGP. According to amino acid substitutions reported, and these variants showed enhanced competition with DAK (Figure 2F). The best variant, AGP-M3, successfully increased starch synthesis of DHA approximately sixfold (Figure 2G).

By using these three engineered enzymes (FLS-M3, FBP-AGR, and AGP-M3), the research team constructed ASAP 2.0, which produced approximately 230 MgL-1 amylose from 20 mM methanol in 10 hours. Compared with ASAP 1.0, ASAP 2.0 increased starch productivity by 7.6 times.

                                                                       Figure 2. Bottleneck problem resolution in ASAP

[Improved enzymatic process, further advanced ASAP 2.0]

Following the above success in ASAP 2.0, the research team combined the enzymatic process with CO2 reduction through a previously developed inorganic catalyst zno-Zro2 to synthesize starch from CO2 and hydrogen. Due to unfavorable conditions of CO2 hydrogenation, the research team developed a chemical enzymatic cascade system with chemical reaction units and enzymatic reaction units in ASAP 3.0. To meet the FLS 'demand for high concentrations of formaldehyde and avoid its toxicity to other enzymes, they further operated the enzyme stimulating unit in two steps (Figure 3A). To synthesize amylopectin from CO2, the research team introduced amylopectin branching enzyme (SBE) from Vibrio vulnificus into ASAP 3.1. This setup produced approximately 1.3 GL-1 amylopectin within 4 hours (Figure 3A). Synthetic amylopectin showed a reddish brown color after iodine treatment, and its maximum absorption was comparable to that of standard amylopectin (FIG. 3B). Both synthetic amylose and amylopectin exhibit the same 1 to 6 proton NMR signals as their standard counterparts (Figs. 3C, 3D).

                                                                                          Figure 3. Starch synthesis from CO2 via ASAP

【 summary 】

This paper corresponding author yan-he ma researcher said that the results from the carbon dioxide to the starch production of industrial workshop manufacturing open a window, if the future cost can reduce the system process to the economic feasibility, compared with agricultural will save more than 90% of the arable land and freshwater resources, avoid pesticide, chemical fertilizer, such as the negative effect on the environment, improve the level of human food security, Promote carbon-neutral bioeconomic development and promote the formation of a sustainable bio-based society.