Biotransformation pathway and side reaction engineering for the whole-cell production of glycolic acid from formaldehyde

doi:10.21203/rs.3.rs-1805701/v1

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Biotransformation pathway and side reaction engineering for the whole-cell production of glycolic acid from formaldehyde

https://doi.org/10.21203/rs.3.rs-1805701/v1

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A very simple biocatalytic system for the synthesis of the industrially relevant C2 chemicals (e.g., glycolic acid (3)) from formaldehyde (1) was established by whole-cell biocatalyst. The biocatalytic system consisted of an engineered glyoxylate carboligase from Escherichia coli K-12 (EcGCL) and an aldehyde dehydrogenase (e.g., AldA from E. coli K-12). To improve glycolic acid production, we have investigated metabolic engineering to regulate substrate consumption and product bypass and E. coli-based strain optimization. The target compound was produced at rates of up to 20.3 U/g dry cells with conversions up to 92%. This study will ensure the sustainability of the green energy industry by synthesizing valuable C2 chemical from a single carbon compound, formaldehyde.

Whole-cell biocatalyst

glyoxylate carboligase

glycolaldehyde

glycolic acid

Escherichia coli

One-carbon compounds are promising feedstocks because of its abundance in nature, non-food resources and potential for sustainability¹. Enzymatic valorization of formaldehyde with high reactivity produces C2 alcohol and acid², and multi-carbon compounds such dihydroxyacetone (C3)³ and erythrulose(C4),⁴ in line with the global trend towards more sustainable and eco-friendly resources (Scheme. S1). Glycolic acid has a variety of skin-related applications in the cosmetic/pharmaceutical industry.⁵ Polymerization of glycolic acid or with other organic acids yields thermoplastic resins for biomedical applications.⁶ Glycolic acid is not just a valuable industrial chemical, but it can also be utilized as a carbon source for biomass.

Currently, since relatively high price of glycolic acid and ethylene glycol in the market, glycolic acid is produced chemically from fossil resources mainly in a process where formaldehyde is carbonylated by treated with water and carbon monoxide or carbonylated by synthesis gas.⁷ Also, it could be produced by microbial-derived enzymatic glycolonitrile conversion⁸ or ethylene glycol bioconversion using Gluconobacter oxidans⁹ as a biocatalyst. However, these toxic chemicals have been used in these chemo-enzymatic processes, therefore their applications are limited. Alternatively, several emerging ideas for the bioproduction from renewable carbon sources(i.e., hexose, pentose sugar) using microbial cell factories were recently reported.¹⁰ However, since the substrate used is the main carbon source in metabolic pathway, these approaches are carbon loss pathway that produce byproducts and these pathways still confront some metabolic hurdles, such as redox imbalance and bypassing the central carbon metabolism.¹¹

A number of synthetic linear pathways have been reported for the production of C2 compounds from formaldehyde. One of the representative pathways is HACL pathway reported in Nat. Metab. (Scheme S1)^{2, 12, 13}. The key reactions are the formation of formyl-CoA from formate and the formation of C2 compound by combining with exogenous formaldehyde. However, regulating the balance between the formyl-CoA synthesis and the reaction with formaldehyde is challenging, resulting in by-products and low yield. Another approach is to produce glycolaldehyde from formaldehyde using carboligase^{3, 4}. One molecule of glycolaldehyde could be biosynthesized from two molecules of formaldehyde using formolase. Formolase, ^{3, 4} on the other hand, has a predominant reaction activity to produce DHA by catalyzing carboligation of three formaldehyde molecules. Also, formolase mutant selective glycolaldehyde reported mutant. Lu et al reported glycolaldehyde synthase (GALS)¹⁴, which catalyzes the condensation of 2 molecules of formaldehyde in to 1 molecule of glycolaldehyde. There have not been studies on the biosynthesis of glycolic acid using this enzyme from formaldehyde yet.

Here, we have designed the simple and linear 2-step pathway to glycolic acid from formaldehyde via glycolaldehyde. (Scheme 1) Also, an E. coli-based whole-cell biocatalyst was constructed based on the novel biosynthesis pathway, and a study was conducted to improve the productivity and glycolic acid production yield of the whole-cell biocatalyst based on metabolic engineering. Therefore, glycolic acid could be synthesized from formaldehyde at a high rate of 20.3 U/g dry cells using an E. coli JM109 (DE3)-based biocatalyst. To biosynthesize glycolic acid via glycolaldehyde, a two-step biosynthetic pathway was developed.

Bacterial strains and cultivation media

Recombinant Escherichia coli BL21(DE3) were inoculated into a Lysogeny broth (LB) medium supplemented with appropriate antibiotics (Supplementary Information, Table S1) for seed cultivation and main cultivation. The expression of genes was induced by adding 0.05 mM isopropyl β-d-thiogalactopyranoside (IPTG) into the cultivation medium to facilitate a soluble expression in E. coli.

Chemicals and reagents

Formaldehyde solution, paraformaldehyde, glycolaldehyde dimer, thiamine diphosphate (ThDP), magnesium chloride hexahydrate, diphenyl amine, sulfuric acid, 1,3-propanediol, O-(2,3,4,5,6-pentafluorobenzyl) hydroxylamine hydrochloride (PFBHA), glycerol dehydrogenase and antibiotics were purchased from Sigma (St. Louis, MO, USA). Phosphate-buffered saline (PBS) and potassium phosphate dibasic anhydrous were obtained from MP biomedicals (CA, USA). N, O-Bis(trimethyl-silyl) trifluoroacetamide (BSTFA), acetylacetone, flavin adenine dinucleotide (FAD), and β-nicotinamide adenine dinucleotide disodium salt hydrate, reduced form (NADH) was obtained from Tokyo Chemical Industry Co. (Tokyo, Japan). Ni-NTA agarose was purchased from Qiagen (Hilden, Germany). Ethyl acetate, ammonium acetate, n-butyl alcohol, potassium phosphate monobasic, and acetic acid glacial were purchased from Duksan Pure Chemical Co. (Ansan, Korea). Cyclohexanone was obtained from Samchun Pure Chemical Co. (Seoul, Korea). LB and IPTG were purchased from Duchefa (RV Haarlem, Netherlands).

Gene cloning and construction of recombinant plasmids

The gene coding for EcGCL from E. coli K-12 and variants were constructed as previously¹⁵. The AldA from E. coli K-12 was amplified by PCR from chromosomal DNA of E. coli K-12 using the primers which were designed for In-Fusion cloning protocol (Takara Bio. Inc, CA, USA). The amplified gene was inserted into the linearized E. coli expression vector pACYCduet-1 (Novagen, Madison, WI) and ligated with the In-Fusion enzyme (Table S2).

CRISPR-Cas9 mediated gene deletion

All constructs including the gRNA, primer and N₂₀ sequences used in this study are given Table S1. To construct gRNA plasmid, the 20bp sequence specific for each target site was synthesized in primers and added into pTargetF plasmid¹⁶ (Table S2). The DNA fragments for deletion of frmA and glcD genes were amplified and those were fused by overlap-extension PCR. All primers were purchased from Cosmo Genetech (Seoul, Republic of Korea). E. coli BL21(DE3) and JM109(DE3) expressing pCas were induced by 50mM arabinose for the induction of λ-red recombinase¹⁷. E. coli electrocompetent cells harboring pCas were mixed with 200 ng of pTargetF and 400 ng of editing template and transformed by electroporation. To cure pTargetF, the deletion strains harboring both pCas and pTargetF plasmid were induced by adding IPTG 1mM. pCas9 was cured by growing the colonies overnight at 37°C non- selectively.¹⁸

Enzyme purification and activity assay

The recombinant cells were harvested by centrifugation at 8,660g for 20 min at 4 °C. The cell pellet was resuspended in lysis buffer [20 mM Tris-HCl (pH 7.5), 300 mM NaCl, and 1mM β -mercapto ethanol (β-ME)], containing 1 mM phenylmethylsulfonyl fluoride (PMSF), and disrupted by sonication. The supernatant was collected by centrifugation at 8,660 g for 30 min at 4 °C to remove the cell debris. The soluble protein sample was loaded onto a nickel affinity column (Qiagen). The bound protein was eluted by a step gradient of imidazole in the lysis buffer. The eluted protein was concentrated and dialyzed against lysis buffer by ultrafiltration with an Amicon Ultra centrifugal filter device (Millipore, USA) with a 30 kDa molecular-weight cutoff. The glycolaldehyde oxidation reaction kinetics was determined by measuring the reaction rates at 30 ℃ by monitoring the increase in Abs₃₄₀. The enzyme concentration was adjusted to 10 μg/mL in 50 mM potassium phosphate buffer (pH 7.0) containing 1 mM NAD⁺ in 100 μL final volume. The reaction was initiated by the addition of varying concentrations of formaldehyde and glycolaldehyde from 0.1 to 10 mM.

Whole-cell biocatalysis

The whole-cell conversion by the recombinant E. coli and its frmA, glcD deletion strains, expressing the EcGCL_R484MN283QL478M and EcAldA were initiated by adding formaldehyde into a 50 mM potassium phosphate buffer (pH 7.0), including the recombinant cells (cell density: 10.8 g dry cells/L). The shaking speed and temperature for reaction were 300 rpm and 40 °C, respectively. After cultivation at 37 ℃ and gene expression at 18 ℃ overnight, the cells were harvested by centrifugation at 3,500 rpm (2,602 g) for 15 min at 4 °C

Reactant analysis

The concentrations of reaction substrates and products were determined as previously.¹⁵ Briefly, The formaldehyde concentrations were assayed by a modification of the Nash reaction for 96-well plate¹⁹. A 100 µL of diluted sample and 100 µL of Nash reagent (0.2 % 2,4-pentanedione, 0.1 M acetic acid, and 3.89 M ammonium acetate) was incubated at 37 °C for 30 min. The concentration was determined by monitoring in Abs₄₁₂. The glycolaldehyde concentrations were determined as previously described²⁰. After addition of the internal standard (cyclohexanone) and O-2,3,4,5,6-pentafluorobenzyl hydroxylamine hydrochloride (PFBHA) solution, the sample was extracted by ethyl acetate. The 80 μL of supernatant was evaporated and 25 μL of BSTFA was added at 65 ℃ for 30 min for derivatization. After derivatization, the samples were analysed by GC/MS. The GC/MS analyses were performed with a model 7820A GC (Agilent) with a HP-5ms fused silica capillary column (30 m length, 0.25 mm film thickness, Agilent Technologies, Palo Alto, CA, USA). For the analysis of glycolic acid concentrations, 300 μL of ethyl acetate and 100 μL saturated sodium chloride as an extraction solvent, containing 20 μL of internal standard (8400 mg/l, 1,3-propanediol in acetonitrile) was added to 20 μL of sample which was prepared by addition of 50 % HCl. The samples were vortexed for a minute and then centrifuged for 2 min at 13,000 rpm. The 250 μL of supernatant was evaporated and 50 μL of BSTFA-TMCS and 10 μL pyridine were added at 65 ℃ for 30 min for derivatization. After derivatization, the samples were analyzed by GC/MS described above.

Biotransformation of formaldehyde into glycolic acid

The biotransformation pathway for the production of glycolic acid from formaldehyde was first designed. Two molecules of formaldehyde are condensed into one molecule of glycolaldehyde by a carboligase, which is further transformed into glycolic acid by an aldehyde dehydrogenase (Scheme 1). A number of carboligases including formolase^{3, 21} and glyoxylate carboligase were examined for the condensation of two molecules of formaldehyde into glycolaldehyde¹⁵. A formolase variant (i.e., FLSH29DQ113C)²¹ was better than parent enzyme in terms of the production yield of glycolaldehyde from formaldehyde. However, the enzyme activity was significantly lower than that of the triple mutant of glyoxylate carboligase from E. coli (EcGCL_R484MN283QL478M¹⁵). Thereby, the EcGCL_ R484MN283QL478M was selected as a candidate for the first step reaction.

The lactaldehyde dehydrogenase (AldA) of E. coli K-12²² and the α-ketoglutaric semialdehyde dehydrogenase (KGSADH) from Azospirillum brasilense²³were investigated for the oxidation of glycolaldehyde into glycolic acid (Scheme 1). The kinetic studies revealed that EcAldA had the greater catalytic efficiency for glycolaldehyde without oxidation for formaldehyde (Table 1). Thereby, EcAldA would be more appropriate for the biotransformation of formaldehyde into glycolic acid.

The two-step biotransformation pathway was examined by conducting the bioconversion of formaldehyde into glycolic acid using the purified EcGCL_ R484MN283QL478M and EcAldA. The reaction was initiated by adding 25 mM formaldehyde into the 50 mM potassium phosphate buffer (pH 7.0) containing 2 mg/mL EcGCL_ R484MN283QL478M. The GC/MS analysis revealed that glycolic acid was produced to 7.6 mM in the reaction medium at t = 1 h (Figure. S1), indicating that first step proceeded to a rate of 9.6 mM/h (Figure. 1).

Table 1. Kinetic parameters of AldA and KGSADH

	Substrate	K_M (mM)	k_cat (s^-1)	k_cat /K_M (mM^-1∙s^-1)
EcAldA	Formaldehyde	n.d.^a	n.d.	n.d.
EcAldA	Glycolaldehyde	0.046	2.03	44.1
AbKGSADH	Formaldehyde	10.3	0.6	0.06
AbKGSADH	Glycolaldehyde	4.0	24.4	6.1

n.d.^a: no detectable activity was observed.

Addition of EcAldA into the reaction medium led to formation of glycolic acid to a rate of 10.7 mM/h. Ultimately, the target product accumulated to 7.5 mM in the reaction medium. This indicated that the biotransformation pathway including the EcGCL_ R484MN283QL478M and EcAldA enzymes could be used for the production of glycolic acid from formaldehyde.

Construction of E. coli based whole-cell biocatalysts system

A whole-cell biocatalyst for the production of glycolic acid was constructed to reach high productivities and high product concentrations. Cell-free enzymatic reactions were hampered by the designed pathways that required cofactor regeneration and toxicity of compounds critical to enzyme activity. In a cascade reaction, formaldehyde is converted to glycolic acid via glycolaldehyde (Scheme 1). The first step's product, glycolaldehyde, was generated at a high rate, and because glycolaldehyde was a fatal intermediate,²⁴ it was swiftly transformed to glycolic acid without accumulation. E. coli-based in vivo cascade system was developed in a stepwise manner. EcGCL_ R484MN283QL478M was overexpressed, and EcAldA had a high catalytic efficiency, therefore EcGCL_ R484MN283QL478M was cloned and expressed in a high-copy number vector (pET21a), while EcAldA was expressed in a low-copy number vector (pACYCDuet). EcGCL_ R484MN283QL478M and EcAldA were expressed to a soluble form in the recombinant E. coli BL21(DE3) p.LIC B3- EcGCL_ R484MN283QL478M, pACYC-EcAldA as designed (Figure. S2). When 50mM formaldehyde was added into the reaction medium containing the whole-cell biocatalysts, formaldehyde converted into glycolic acid over half. The biotransformation led to formation of glycolic acid to 16.0 mM without accumulation of glycolaldehyde below 2 mM. The reaction rate reached 22.3 U/g dry cells at t < 0.5 h, and production rate decreased after 1 h with loss of enzymatic activity. The increase of formaldehyde concentration to 75 mM in the reaction medium did not lead to enhancement in the glycolic acid concentrations. Formaldehyde was completely consumed within 4 h, but the desired product, glycolic acid, was not sufficiently produced and glycolaldehyde also accumulated. (Figure. 2) The initial reaction rate was diminished to 7.8 U/g dry cells (Table 2), and it is assumed that EcGCL_R484MN283QL478M rapidly lost activity owing to formaldehyde toxicity toward enzymes and E. coli cells, and that as glycolaldehyde accumulated, EcAldA lost activity and the reaction was ceased. The formaldehyde detoxification pathway and the glyoxylate shunt are hypothesized to be closely related to formaldehyde consumption. E. coli has a very sensitive fine-tuned regulatory mechanism that dissolves formaldehyde into CO₂. Glycolic acid is converted to glyoxylic acid, which flows into the TCA cycle via the glyoxylate shunt. This level might rise as the substrate concentration increases, and despite the completely consumption of formaldehyde, very little glycolic acid (14.4 %) was produced. The flux into our designed pathway could be improved by blocking these side reactions with metabolic engineering.

Table 2. The effect of substrate concentration on recombinant Escherichia coli BL21(DE3) activity and conversion yield of glycolic acid.

Formaldehyde concentration

(mM)

Specific activity ^a

(mol/g cell/min)

Conversion yield^b (%)

21.3

45.6

7.8

14.4

^a The biotransformation rates were calculated with the cell and product concentrations at t = 0.5 h. The product concentrations were measured by GC/MS analysis.

^b The conversions were estimated with the concentrations of substrates added and products produced during the biotransformations shown in Figure 2. The substrate and product concentrations were measured by GC/MS analysis.

Improvement of whole-cell biocatalyst in engineered E. coli

Metabolic engineering of E. coli for the production of glycolic acid from glycolaldehyde

To improve the productivity of glycolic acid from formaldehyde, an engineered strain of E. coli based on BL21 (DE3) was constructed. In E. coli, the major pathway involved in formaldehyde detoxification is GSH-dependent and composed of GD-Faldh (encoded by frmA) and Fgh ( encoded by frmB and yeig)²⁵. (Scheme 2) YeiG was continuously expressed in E. coli, but FrmB was activated by exogenous formaldehyde (0.25 mM) and was reported to be expressed 45-fold, which might explain the low yield²⁶. Also, glycolic acid is converted to glyoxylic acid by the glcDEF complex and enters the TCA cycle through the glyoxylate shunt²⁷, which can be used as a carbon source in the cell (Scheme 2). We constructed an engineered host that knocked out performance-inhibiting pathways from formaldehyde oxidation (∆frmA) and glycolic acid conversion (∆glcD).

Catalytic performance of E. coli deletion variants

To evaluate the whole-cell biotransformation performance of engineered strains (E. coli BL21(DE3) ΔfrmA, ΔglcD and ΔfrmAΔglcD), EcGCL and EcAldA were expressed in knockout strains and constructed as a biocatalyst to produce glycolic acid from formaldehyde. A biocatalyst using E. coli BL21 (DE3) wild type consumed formaldehyde within 4 h. The glycolic acid concentration was up to 18.5 mM and 73.8 % when a biocatalyst using frmA reacted with formaldehyde at a 50 mM concentration. It is assumed that EcGCL lost its activity because of the residual formaldehyde toxicity since the rate at which formaldehyde was consumed decreased significantly after 2 h and the formaldehyde detoxification path was blocked. (Figure. 3A, Table. 3) In biocatalyst using ΔglcD, the reaction rate was comparable to that of wild type, and formaldehyde decreased after 2 h, but glycolic acid almost halted the reaction after 2 h, and the conversion yield was 55%, which increased by 1.2-fold compared to wild type (Figure. 3B). In terms of overall reaction performance, biocatalyst using ΔfrmA ΔglcD (Figure. 3C) showed a similar pattern to biocatalyst using frmA and produced the most, with 75 % glycolic acid conversion in 4 h. This implies that the effect of the formaldehyde detoxification pathway via frmAB in BL21(DE3) has the greatest influence on the overall response profile. In addition, in the biocatalyst using ΔfrmA ΔglcD, the initial reaction rate was increased to 34.0 U/g dry cells (Table. 3), and 90% of the substrate consumption was converted to glycolic acid without glycolaldehyde accumulation. This result shows that the designed pathway was improved through metabolic engineering.

Biocatalyst engineering for production of glycolic acid

Evaluation of additional E. coli strains for improving the production of glycolic acid

For converting high-concentration substrates, the performance of the E. coli B strain, which is generally reported to be more favourable for protein expression, and the E. coli K-12 strain, which demonstrated more diversified intracellular responses to overcome stress, were evaluated. In case of reaction with addition of formaldehyde 50mM, JM109(DE3)-based strain reached 20.3 U/g dry cells at t < 0.5 h. The final product concentration was 23.1 mM without the accumulation of intermediates,

Table 3. The effect of recombinant E. coli BL21(DE3) deletion variants on activity and conversion yield of glycolic acid.

Strain	Specific activity ^a (U/g CDW)	Conversion yield ^b (%)
BL21(DE3)	21.3	44.8
BL21(DE3) △frmA	15.6	73.8
BL21(DE3) △glcD	35.5	55.2
BL21(DE3) △frmA△glcD	35.8	75.2

^a The biotransformation rates were calculated with the cell and product concentrations at t = 0.5 h. The product concentrations were measured by GC/MS analysis.

^b The conversions were estimated with the concentrations of substrates added and products produced during the biotransformations shown in Figure 3. The substrate and product concentrations were measured by GC/MS analysis.

and glycolic acid was produced up to 91.6 % of the theoretical yield (Figure 4). This result was comparable to the biotransformation result reported in Nat. Mebol.² of yielding more than 10mM of glycolic acid after 24 hr by adding 25mM substrate in the system developed using HACL. (Figure. 4A) The BL21(DE3) based strain showed a significantly decreased production rate after 1 hour, but JM109(DE3) accumulated glycolic acid at a high rate even after 2 h. It demonstrates that a mass balance was established with substrate consumption and product yield, resulting in higher glycolic acid synthesis. JM109(DE3) reaction performance decreased with increasing formaldehyde concentration, but it still demonstrated higher initial rates and conversion rates than BL21(DE3) and ER2566. (Figure. 4, Figure. S3)

Genes involved in the glyoxylate shunt and tricarboxylic acid (TCA) cycle were up-regulated in the K strain as a result of analysing the transcriptomes of strains K and B in the previous study^{28, 29}. In BL21, however, both the glyoxylate shunt and the TCA cycle are activated. TCA cycle activity needs to be optimized between stress responses and redox balance, and low TCA cycle activity in JM109 can lead to a decrease in intracellular NADH levels, leading to an increase in glycolic acid production through our designed NAD⁺-dependent pathway.³⁰ Acetyl-coA synthase (acs) is also transcribed from BL21 and converted to acetyl CoA when acetic acid increases, but it is not transcribed in JM109, suggesting that it leads to acetic acid accumulation.³⁰ As a result of this research, the JM109 strain had a comparable expression pattern to the BL21 strain (Figure. S2) and produced a high yield of glycolic acid. The key reason is that formaldehyde is not converted to the bypass pathway but mainly goes to the desired pathway. Therefore, a biocatalyst using JM109(DE3) is suitable as a host cell for further experiments.

The JM109(DE3) strain includes an incomplete glyoxylate shunt that converts glycolic acid into a carbon source, and its formaldehyde consumption/product (e.g., glycolaldehyde, glycolic acid) production balance is comparable to that of BL21(DE3). It is assumed that this is due to a decrease in the gene expression level of the enzyme involved in the formaldehyde detoxification pathway. Further studies on omics such as transcriptome and proteome of formaldehyde metabolism in E. coli strains are needed. Overall, glycolic acid was produced from formaldehyde in a simple and linear manner using an E. coli-based biocatalyst, and a bioconversion system was constructed that produced glycolic acid at a high rate and yield by host cell engineering.

This study demonstrated for the first time that the biosynthesis of formaldehyde into glycolic acid via glycolaldehyde in EcGCL-based whole-cell biocatalyst in a simple two-step process. Side reaction engineering improved glycolic acid yield and productivity by metabolic engineering and optimization the host strain. This study will contribute to development of an engineered whole-cell biocatalyst for valorization of one-carbon chemicals in a sustainable manner.

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Schemes 1-2 are available in the Supplementary Files section.

There is NO Competing Interest.

SINat.Comm.docx
Supplementary information
Scheme1.png
Scheme 1. C1 cascade conversion pathway. Formaldehyde (1) is converted into glycolic acid (3) via glycolaldehyde (2).
Scheme2.png
Scheme 2. Schematic view of glycolic acid production system by engineered E. coli whole-cell biocatalyst.

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Biotransformation pathway and side reaction engineering for the whole-cell production of glycolic acid from formaldehyde

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