Formic acid biotransformation engineering strain and application thereof

Abstract

The invention relates to the technical field of biology, in particular to the technical field of formic acid biological utilization and efficient biological conversion. The invention provides a formic acid biotransformation engineering strain. The bacterial strain is obtained after a plasmid pED31-FFM and a plasmid pAD31-gcvTHP are introduced into escherichia coli, and the plasmid pED31-FF M is obtained after an ftfl-fchA-mtdA (FFM) gene is connected to a pED31 empty plasmid; the plasmid pA D31-gcvTHP is obtained by connecting a gcvTHP gene to a pAD31 empty plasmid, and the plasmid pA D31-gcvTHP can be obtained by connecting the gcvTHP gene to the pAD31 empty plasmid. According to the strain provided by the invention, under the initial concentration of 20-60mM formic acid, the multiplication time is within 5 hours, the biomass yield can reach 6g CDW / mol formic acid, the single-cell protein yield can reach 2.45-4.2 g / mol, and the international leading level of formic acid conversion and biomass is reached.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and particularly to the field of formic acid bioutilization and efficient biotransformation technology. Background Technology

[0002] Using renewable electricity to reduce CO2 into one-carbon compounds such as formic acid, formaldehyde, and methanol, and then coupling this with microbial utilization to convert it into chemicals, is one of the important strategies for achieving sustainable recycling of carbon resources and the development of green industries.

[0003] Formic acid is the easiest water-soluble one-carbon compound to prepare, exhibiting high solubility, low toxicity, mature technology, high economic and technical feasibility, good bioavailability, high conversion rate, and non-flammability. Compared to methanol, its electrocatalytic production offers better electronic economics. It is also safer to transport and store, and exhibits higher safety during microbial cultivation. Developing formic acid-efficient strains and using formic acid as a carbon source to replace traditional industrial microbial glycosyl raw materials is an effective means to reduce carbon emissions and improve green bioproduction routes.

[0004] Natural formic acid-utilizing strains mainly include acetic acid-producing bacteria, such as *Acetobacterium woodii*, *Clostridium ljungdahlii*, and *Moorellathermoacetica*; methanogenic bacteria, such as the hydrotrophic methanococcus maripaludis; sulfate-reducing bacteria, such as *Desulfovibrio vulgaris*, *Desulfovibrio baarsii*, *Desulfovibrio desulfuricans*, and *Desulfarculus baarsii*; and other microorganisms that utilize formic acid by oxidizing it with formic acid dehydrogenases to provide reducing power for CO2. Natural marine sodium-dependent *Vibrio natriegens* exhibits strong formic acid tolerance (60 g / L) and metabolic capacity, showing great potential for formic acid bioavailability. However, this strain cannot currently utilize formic acid as its sole carbon source for growth and metabolism, and its formic acid assimilation rate needs improvement. Research on most natural formic acid-utilizing strains is still incomplete, with limited genetic modification tools, making them unsuitable for metabolic engineering and industrial production. In contrast, research on model microorganisms such as E. coli and S. cerevisiae is more comprehensive. Constructing highly efficient formic acid-utilizing strains is not only more suitable for metabolic engineering and industrial production but also more conducive to the further development of the formic acid bioeconomy.

[0005] Discovered and optimized formate assimilation pathways include: the serine cycle, the modified serine cycle, the reduced acetyl-CoA pathway (Wood-Ljungdahl pathway, WL pathway), the reduced glycine pathway (rGlyP), the formate-formaldehyde-RuMP pathway, and the formate-formyl-CoA-serine pathway. Among these, the WL pathway is a known natural formate assimilation pathway with fewer reaction steps and lower ATP consumption, but heterologous expression of this pathway is challenging. The rGlyP pathway is short (5 enzymes), has low energy (2 ATP) and reducing power (3 NAD(P)H) requirements, minimal interference with central metabolic pathways, and good oxygen tolerance, making it a promising carbon fixation pathway. It has been successfully constructed in *E. coli* and *Saccharomyces cerevisiae*, exhibiting high formate tolerance (750 mM) in *Saccharomyces cerevisiae*. However, the formate utilization efficiency of the reconstructed *E. coli* and yeast strains remains low, with the doubling time of engineered strains using formate as the sole carbon source exceeding 6 hours. Using the reduced glycine pathway, 1.2 mM lactate was produced in *E. coli* by coupling LDH (maximum yield 10%); the PHB pathway was introduced, and PHB production was observed in *E. coli*. Because current formic acid-producing strains still suffer from long growth cycles, weak formic acid utilization, and low assimilation rates, there is an urgent need to develop strains that efficiently transform formic acid. Summary of the Invention

[0006] In view of this, the present invention provides a highly efficient formic acid biotransformation engineered strain. This strain is obtained by introducing plasmids pED31-FFM and pAD31-gcvTHP into *Escherichia coli*. Plasmid pED31-FFM is obtained by ligating the ftfl-fchA-mtdA (FFM) gene to an empty pED31 plasmid; plasmid pAD31-gcvTHP is obtained by ligating the gcvTHP gene to an empty pAD31 plasmid.

[0007] Furthermore, the *E. coli* was obtained through one or more of the following genetic engineering operations:

[0008] (1) Knock out the sdaB gene in wild-type Escherichia coli;

[0009] (2) The Escherichia coli sdaA gene was integrated into the sdaB gene position of strain ΔsdaB.

[0010] (3) Integrating formate dehydrogenase PseFDH into the Escherichia coli genome;

[0011] (4) Gene mutations were performed on engineered Escherichia coli, with mutation sites including RpoB (I854N), Pc nB (V102E), and ProA (A383E).

[0012] (5) Gene mutations were performed on engineered Escherichia coli, with mutation sites including: gcvR(P(-45)Ins T), acs(Δ1915-1918), Pat(L206P);

[0013] (6) Gene mutation of engineered Escherichia coli, with mutation sites including: ArcA (E94*) and ActP (A350T).

[0014] The present invention also provides the application of the formic acid biotransformation engineered strain in the utilization of formic acid.

[0015] The present invention further provides the application of the engineered strain in the production of single-cell proteins. Attached Figure Description

[0016] Figure 1 This is a diagram of the construction of plasmid pED31-FFM.

[0017] Figure 2 This is a diagram showing the construction of plasmid pAD31-gcvTHP. Detailed Implementation

[0018] Example

[0019] To obtain highly efficient formic acid biotransformation strains, we rationally designed and combined targeted domestication methods under the premise of high assimilation and weak dissimilation, resulting in formic acid biotransformation strains with rapid biotransformation and high biomass yield. The biomass yield was 6 g CDW / mol formic acid. The single-cell protein yield reached 2.45-4.2 g / mol formic acid.

[0020] Advantages of this invention:

[0021] 1. Achieving efficient formic acid conversion using formic acid as the sole carbon source: This provides an efficient formic acid bioconversion engineered bacterium that can grow using formic acid as the sole carbon and energy source.

[0022] 2. Enhanced formic acid biotransformation pathway: Overexpression of the reduced glycine pathway (rGly P) via plasmid significantly enhanced formic acid assimilation. This pathway, combined with activation of the TCA cycle, further optimized the metabolic transformation of formic acid.

[0023] 3. Activation and Global Regulation of the TCA Cycle: Through the engineering of global regulatory elements, the inactivation of Ar cA inhibitors significantly activated the TCA cycle, enabling the strain to maintain efficient growth under low-carbon and low-energy conditions. This optimization not only reduced the accumulation of acetic acid byproducts but also enhanced the metabolic mobility and energy utilization efficiency of the strain.

[0024] 4. Rapid growth and high biomass yield: At an initial concentration of 20-60 mM formic acid, this strain achieves a doubling time of no more than 5 hours using formic acid as the sole carbon source, with a biomass yield reaching 6 g CDW / mol formic acid. This level represents an internationally leading position in the field of formic acid conversion and biomass production.

[0025] 5. High-efficiency production of single-cell protein using formic acid as raw material: The formic acid biotransformation engineered strain provided by this invention can achieve a single-cell protein yield of 2.45-4.2 g / mol formic acid at an initial formic acid concentration of 20-60 mM, demonstrating excellent potential for single-cell protein production.

[0026] Example 1

[0027] Construction of plasmid pED31-FFM / pAD31-gcvTHP

[0028] The genetically optimized formate tetrahydrofolate ligase-methylenetetrahydrofolate cyclohydrolase-methylenetetrahydrofolate dehydrogenase gene ftfl-fchA-mtdA (FFM), containing the complete reading frame of the pTac promoter and RBS sequence, was ligated into the pED31 empty plasmid via NdeI and XhoI restriction enzyme digestion, transformed into DH5α, cultured in an incubator, and subsequently validated by colony PCR. Positive clones were then sent for sequencing.

[0029] The gcvTHP gene from E. coli was synthesized, containing the complete reading frame of the pLacO1 promoter and RBS sequence. It was ligated into the pAD31 empty plasmid by EcoNI and SalI restriction enzyme digestion, transformed into DH5α, cultured in an incubator, and then verified by colony PCR. Positive clones were sent for sequencing.

[0030] Example 2

[0031] Methods for constructing strains

[0032] 1. CRISPR-Cas9 gene knockout and integration method

[0033] The CRISPR-Cas9 method was used to knock out and integrate E. coli BW25113 into the genome. The specific operation steps are explained using BW25113ΔmetE as an example.

[0034] (1) pCas9 plasmid was transformed into BW25113 to obtain BW25113pCas9 single clones. Single clones were picked and cultured overnight at 30℃. The inoculum was then transferred to fresh LB medium at a 1:100 ratio, and 10% 1M L-ara was added for induction culture for 2 hours until OD. 600 Approximately 0.6, prepared as competent cells for later use.

[0035] (2) During step (1), prepare the fragment. Amplify 500 bp each of the upstream and downstream fragments of the metE gene, and then design appropriate primers to connect the upstream and downstream fragments to obtain the up-metE-down fragment.

[0036] (3) Design a suitable N20 for metE using online gRNA-N20, and synthesize primer pTraget-metE-N20. Amplify with pTraget-metE-N20 and pTarget-R, transform into DH5α, select single clones for sequencing, and obtain the correct plasmid pTraget-metE-N20 for later use.

[0037] (4) Transform up-metE-down and pTraget-metE-N20 into BW25113pCas9, culture overnight in a 30℃ biochemical incubator, pick single clones, and perform colony PCR verification. The gene knockout should be consistent with the size of the overlap fragment and shorter than the wild-type strain fragment. If the verification is correct, the target strain is obtained.

[0038] (5) Induce expression of the correct single-clone strain with 0.3mM IPTG and culture for about 12 hours. Then streak the strain in three regions on a plate. Select single clones to verify the loss of pTarget-metE-N20 and perform a second colony PCR verification to obtain single clones that have lost the plasmid but have correct colony PCR results.

[0039] (6) Select single clones of the bacteria that lost N20, incubate them overnight at 42°C to verify the loss of pCas9, and obtain the antibiotic-free target strain BW25113ΔmetE.

[0040] 2. By constructing auxotrophic strains, a formic acid assimilation module was built, and a complete formic acid biotransformation pathway was constructed step by step.

[0041] First, the proteins encoding methylenetetrahydrofolate, encoded by the metE and metH genes, were knocked out to construct the methylenetetrahydrofolate-accumulating strain BW25113ΔmetEΔmetH. Based on this, serA and gcvP were knocked out to construct the serine-deficient Escherichia coli BW25113ΔmetEΔmetHΔserAΔgcvP.

[0042] The ftfl-fchA-mtdA gene cluster derived from Methylobacterium extorquens AM1 was screened, and a serine-deficient strain was introduced into the strain using plasmid pED31-FFM. The strain recovered growth in M9M medium containing glycine, formic acid, and glucose, demonstrating the patency of the formic acid-to-methylenetetrahydrofolate synthesis module. The formic acid-to-methylenetetrahydrofolate synthesis module P1 was obtained.

[0043] On the other hand, based on BW25113ΔmetEΔmetH, glyA was knocked out to construct a glycine growth auxotroph strain, BW25113ΔmetEΔmetHΔglyA. Since the reverse reaction of the E. coli endogenous glycine cleavage system could not support the growth of glycine auxotrophic strains, an overexpression plasmid, pAD-gcvTHP, was constructed. The endogenous promoter of gcvTHP was replaced with LacO1 to avoid endogenous regulation by E. coli, and the RBS sequence was optimized. Plasmids containing different RBS strengths were transformed into strains evaluating glycine synthesis capacity, and the growth capacity of the strains was measured. The optimal glycine synthesis plasmid, pAD31-gcvTHP, was obtained and served as module P2 for CO2 fixation and glycine synthesis using methylenetetrahydrofolate.

[0044] Wild-type Escherichia coli can utilize serine as the sole carbon source for growth, and generate pyruvate using the endogenous glyA-encoded serine hydroxymethyltransferase and sdaA and sdaB-encoded serine deaminases. However, the growth is slow. In order to construct a more efficient pyruvate generation module, the overexpression plasmid pAC-sdaA was constructed and transformed into knockout strains for growth testing.

[0045] Overexpression of the sdaA gene accelerated the growth of all strains, with the fastest-growing strain being BW25113ΔsdaB.

[0046] To reduce the burden caused by the presence of plasmids in the strain, the sdaA gene was integrated into the sdaB gene position, thus constructing the serine utilization enhanced strain BW25113sdaB::sdaA.

[0047] Using serine as the sole carbon source, the utilization of this strain showed that its growth was significantly higher than that of the wild-type strain. It was used as a starting strain for subsequent experiments, containing the P3 module for glycine-to-pyruvate synthesis.

[0048] The formate dehydrogenase PseFDH with the highest NADH regeneration activity was screened in 20% CO2 air and integrated as a formate energy supply module into the genome of strain BW25113sdaB::sdaA to obtain strain BW25113sdaB::sdaA fadM::PseFdh. Based on this, formate assimilation modules P1 and P2 were introduced to construct a complete formate-trophic strain BW25113sdaB::sdaA FadM::PseFdh pED31-FFM / pAD31-gcvTHP. This strain BW25113sdaB::sdaA FadM::PseFdh pED31-FFM / pAD31-gcvTHP was labeled as #1.

[0049] 3. Engineered strains with improved formic acid biotransformation efficiency were obtained through laboratory adaptive evolution.

[0050] To improve the bioconversion efficiency of formic acid, a formic acid-trophic *E. coli* strain using formic acid as its sole energy source underwent laboratory adaptive evolution. In the initial generation, 0.2 g / L glycine was added to maintain growth, and the glycine concentration was gradually reduced to 0 g / L with subsequent generations. The initial sodium formate concentration was 40 mM. 600 Once the cells reach approximately 0.4 g / mL, they are passaged, with each generation diluted to 0.08-0.1 g / mL before starting a new culture cycle. Based on strain #1, through evolution, an engineered strain #39 was obtained that can grow entirely dependent on formic acid. This strain has a biomass yield of approximately 6.2 g cell dry weight per mole (g CDW / mol) of formic acid. This strain includes the mutant sites RpoB (I854N), PcnB (V102E), and ProA (A383E), enabling it to grow using formic acid and CO2 as carbon sources, with a maximum OD... 600 It can reach 0.5, with a doubling time of about 30 hours.

[0051] Strain #73 was also obtained, which had additional mutation sites: gcvR(P(-45)InsT), acs(Δ1915-1918), and Pat(L206P). The strain exhibited improved growth performance, with a maximum OD of 0.7 and a doubling time shortened to approximately 10 hours. However, accumulation of intermediate byproducts of acetic acid was observed during the conversion of formic acid.

[0052] 4. Engineering modifications reduce the accumulation of the byproduct acetic acid and improve the bioconversion efficiency of formic acid.

[0053] To address the challenges in acetic acid production, we constructed an engineered strain that inactivated the global inhibitory factor ArcA in the TCA cycle. The DNA-binding domain of the ArcA protein was truncated, rendering it completely inactive. qPCR validation confirmed a comprehensive upregulation of the TCA cycle gene transcription level, directly reducing the generation of intermediate byproducts in acetic acid production by 90%, with no significant difference in strain growth. The resulting engineered strain, FEB-rGlyP-aTCA, further contained the mutant sites ArcA (E94*) and ActP (A350T), achieving a maximum OD of 0.92 and a doubling time shortened to 5 hours. Biomass yield was 3.5-6 g CDW / mol formic acid, and single-cell protein yield reached 2.45-4.2 g protein / mol formic acid.

[0054] Biomass yield is compared using units of formic acid per mole:

[0055] Calculation method:

[0056] Note: E. coli dry weight is 0.31 g / L / OD, formic acid consumption is in moles, and biomass yield is in grams of dry cell weight / mol formic acid (g cell dry weight / mol formic acid, g CDW / mol formic acid).

[0057] Table 1: Growth and Formic Acid Consumption of Formic Acid-Nutritional Escherichia coli

[0058]

[0059]

[0060] Table 2: Formic acid and CO2 as carbon source for single-cell protein production by engineered strain FEB-rGlyP-aTCA

[0061]

[0062] Table 3: List of primers used

[0063]

[0064]

Claims

1. Engineered strains, characterized by: The plasmids pED31-FFM and pAD31-gcvTHP were introduced into E. coli to obtain the following: The plasmid pED31-FFM was obtained by ligating the ftfl-fchA-mtdA(FFM) gene into the empty pED31 plasmid. The plasmid pAD31-gcvTHP was obtained by ligating the gcvTHP gene into the empty pAD31 plasmid.

2. The engineered strain according to claim 1, characterized in that, The *E. coli* was obtained through one or more of the following genetic engineering procedures: (1) Knock out the sdaB gene in wild-type Escherichia coli; (2) The Escherichia coli sdaA gene was integrated into the sdaB gene position of strain ΔsdaB. (3) Integrating formate dehydrogenase PseFDH into the Escherichia coli genome; (4) Gene mutations were performed on engineered Escherichia coli, with mutation sites including RpoB (I854N), Pc nB (V102E), and ProA (A383E). (5) Gene mutations were performed on engineered Escherichia coli, with mutation sites including: gcvR(P(-45)Ins T), acs(Δ1915-1918), Pat(L206P); (6) Gene mutation of engineered Escherichia coli, with mutation sites including: ArcA (E94*) and ActP (A350T).

3. The application of the engineered strain according to any one of claims 1 to 2 in the use of formic acid.

4. The use of the engineered strain according to any one of claims 1 to 2 in the production of single-cell proteins.

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