Analysis of putrescine toxicity on M. alcaliphilum 20Z
In general, putrescine is toxic to microorganisms [18]. Therefore, the impacts of extracellular putrescine inhibition on M. alcaliphilum 20Z growth was first examined. M. alcaliphilum 20Z was cultured to an exponential phase and exposed to different putrescine concentrations of 50 mM, 100 mM, 200 mM, and 400 mM (Fig. 2). M. alcaliphilum 20Z showed significantly reduced growth rates in the presence of 50 mM and higher putrescine concentrations. Approximately, 50 mM of putrescine dihydrochloride (equivalent to 4.4 g/L putrescine) caused cell lysis. Based on this observation, we concluded that low putrescine tolerance could be a main barrier for metabolic engineering of M. alcaliphilum 20Z for putrescine production.
Enhancement of putrescine tolerance of M. alcaliphilum 20Z by adaptive laboratory evolution
Adaptive evolution to improve putrescine tolerance of M. alcaliphilum 20Z was first conducted to overcome low putrescine tolerance of M. alcaliphilum 20Z. Cells exposed to 100 mM of putrescine dihydrochloride in the tolerance test were incubated in NMS with 0.2% methanol at 30 °C, 230 rpm for 15 days. Then, 2 mL of culture broth were transferred to fresh NMS medium with 0.2% methanol to recover. A long lag phase of 3 days was observed, and active cells were transferred to fresh NMS medium. Cells were grown to an OD600 of 0.4 and exposed to 100 mM of putrescine dihydrochloride for 7 days. After 5 replication cycles (approximately more than 65 days in total), a strain with a high tolerance to putrescine was obtained (referred to as the 20ZE strain). The evolved putrescine-tolerant 20ZE strain was able to grow in the presence of 400 mM putrescine dihydrochloride. The growth rate of the evolved strain was comparable to that of wild-type strain (0.089 h−1), and the evolved strain showed even slightly better growth rate in the presence of 100 and 200 mM putrescine (0.121 h−1 and 0.111 h−1, respectively) (Fig. 3). Extension of putrescine tolerance on M. alcaliphilum 20Z allowed the evolved strain to be employed for further metabolic engineering for putrescine production from methane.
Construction of a platform host strain for putrescine production from methane and methanol
Putrescine can be synthesized in vivo via ornithine by constitutive ornithine decarboxylase (speC) or inducible ornithine decarboxylase (speF) and arginine by arginine decarboxylase and agmatinase. M. alcaliphilum 20Z possesses alternative agmatine deiminase, which hydrolyzes agmatine to ammonia and N-carbamoylputrescine. The latter is subsequently hydrolyzed to putrescine, ammonia, and carbon dioxide by N-carbamoylputrescine amidohydrolase [9]. In addition, M. alcaliphilum 20Z also possesses a putrescine utilization pathway, which converts putrescine to spermidine by spermidine synthase (speE). There are five genes predicted as spermidine synthase, of which three genes have lengths of about 516 (MEALZ_1869), 825 (MEALZ_1164), and 856 (MEALZ_3010) amino acids, respectively. The two other genes are very similar to spermidine synthases of E. coli, which have protein lengths of 282 (speE1-MEALZ_3408) and 267 (speE2-MEALZ_3304) amino acids, respectively. Therefore, we tried to inactivate these two genes with similar protein lengths of E. coli spermidine synthase, and test whether putrescine could be accumulated in the mutant M. alcaliphilum 20Z strain. The spermidine synthase gene (speE1) was knocked out through unmarked allelic exchange using a sucrose counter-selection system [19]. Vector pCM433 was constructed with two flanking homology regions upstream and downstream of MEALZ_3408, resulting in vector pCSE1. Vector pCSE1 was introduced into strain 20ZE by electroporation to be integrated into the genome of 20ZE by single crossover. The single-crossover recombinants were transferred to fresh NMS medium containing 2.5% (w/v) of sucrose and were selected for the mutants that excised the vector by PCR using the primer outside the flanking regions. The speE1 mutant was obtained as strain 20ZES1, which accumulated 0.35 mg/L of putrescine after 96 h cultivation. The spermidine concentration in the culture broth was also analyzed, and there was no spermidine accumulation. This means that the knockout of the spermidine synthase (MEALZ_3408) blocked the conversion of putrescine to spermidine. Spermidine and other polyamines are involved in critical physiological processes in bacteria such as cell growth, biofilm formation, stress response, and proliferation [20], but, interestingly, spermidine was proved to be not essential for the growth of E. coli and Saccharomyces cerevisiae [21, 22]. Obviously, inactivation of putrescine degradation and utilization pathway in E. coli resulted in a higher putrescine titer [6]. Similarly, inactivation of the putrescine utilization pathway allowed putrescine to be accumulated in the engineered strain 20ZES1.
To increase putrescine production in the M. alcaliphilum 20Z, ornithine decarboxylase was expressed to directly convert ornithine to putrescine. Constitutive ornithine decarboxylase (speCEc) and a native putrescine transporter (potE) from E. coli K12 were amplified and cloned into an IncP-based broad host-range vector pAWP89 under control of a pTac promoter, resulting in a pACE vector. An ornithine decarboxylase (speCOb) from M. trichosporium OB3b, a type II methanotroph, was also cloned into the pAWP89 vector, resulting in vector pACO. Moreover, the codon adaptation index (CAI) of those genes (for predicting gene expression level) was calculated based on a M. alcaliphilum 20Z codon usage table. The CAI values of speCEc and speCOb were relatively high at 0.73 and 0.78, respectively. Vectors pACE and pACO were successfully transformed into wild-type 20Z by electroporation, resulting in strains 20Z-pACE and 20Z-pACO, respectively. Recombinant strains were cultured in a shake flask containing 50 mL of NMS medium with 50% (v/v) methane. Putrescine accumulation in the supernatant was analyzed by HPLC as described above. After 144 h of incubation, recombinant strain 20Z- pACE produced 2.27 ± 0.42 mg/L of putrescine, while the 20Z-pACO strain produced 12.44 ± 0.86 mg/L of putrescine, approximately five times higher. Enzyme ornithine decarboxylase (speCOb) from M. trichosporium OB3b showed high activity even with an alkali pH in the culture medium. Vector pACO would be used for further putrescine production experiments.
The methylated vector pACO harvested from 20Z strains was easily transformed to strain 20ZES1 with high efficiency, resulting in strain 20ZES1-pACO. Interestingly, strain 20ZES1 produced 18.43 ± 1.08 mg/L putrescine in shake flasks after 144 h (Fig. 4). This production increased by 32.5% compared to the wild-type strain harboring vector pACO.
Metabolic engineering strategy for enhancing putrescine production based on in silico simulation
Genome-scale metabolic model can be employed to identify gene knockout strategies to optimize the metabolic pathway for the target product. Genome-scale metabolic models have successfully been applied to M. alcaliphilum 20Z [14, 16]. OptGene is computationally efficient, and it has been successfully applied (i) in Saccharomyces cerevisiae to improve succinate production by 30-fold [23], (ii) in Synechocystis to allow for growth-coupled biofuel production [24], and (iii) in M. alcaliphilum 20Z for improving the production of 2,3-butandiol [16]. OptGene was performed to identify gene deletion strategies to improve the biomass production-coupled yield (BPCY) as well as the yield of putrescine. In the OptGene algorithm, reaction knockouts were randomly introduced to obtain a mutant population [25]. The objective function was calculated using minimization of metabolic adjustment (MOMA), which identified the closest flux point to the wild-type point making it compatible with the gene deletion constraint [26].
Maximization of the specific growth rate and secretion of putrescine was used as the objective function with flux balance analysis (FBA) to predict the phenotype of the wild-type strain. The maximum theoretical yield and maximum productivity of putrescine production from methane were computed to be 0.579 g/g methane and 0.937 mmol/gDCW/h. Putrescine production is not favorable at the optimal biomass growth rate, as indicated by the FBA using maximization of the specific growth rate as the objective function. This resulted in no putrescine accumulation.
An in silico evolutionary programming-based method was performed using OptGene to optimize putrescine production. OptGene-derived mutations that increased BPCY for putrescine production were generated and are listed in Additional file 1: Table S1. The most common knockouts for putrescine production were acetate kinase (ACKr), serine hydroxymethyltransferase (glyA), and methylenetetrahydrofolate dehydrogenase (MTHFD). GlyA and MTHFD are two genes that evolved in the tetrahydromethanopterin (H4MPT) pathway and serine cycle. Through this pathway, formaldehyde was oxidized to formate, which played a role in formaldehyde detoxification and provided NADH for methane oxidation [27]. Knockout of these two genes was not favorable for strain growth in methane. Moreover, the predicted flux value via the H4MPT pathway was small, i.e., 0.1 compared with 11.6 via the RuMP pathway in M. alcaliphilum 20Z [14]. Knockout of ACKr gave a BPCY of 0.0027 with the highest productivity of 0.66 mmol/gDCW/h of putrescine and a yield of 0.408 g-(putrescine)/g-CH4, which is approximately 70% of the theoretical yield. The maximum specific growth rate was predicted as 0.032 h−1. Thus, it was selected as a knockout target. Subsequently, lactate dehydrogenase (LDH) was also selected as a promising target to enhance putrescine production. With the knockout mutant of ACKr, the flux redistribution toward putrescine formation from acetyl-CoA can be improved, because of the decrease in the flux from acetyl-CoA to acetate.
A triple mutant strain was successfully generated using sucrose counter-selection, resulting in strain 20ZE3. Putrescine production by the mutant strain was examined through the transformation of vector pACO to construct strain 20ZE3-pACO. Strain 20ZE3-pACO was able to accumulate 26.69 ± 1.86 mg/L of putrescine after 144 h of cultivation, which is a 37.03% improvement compared to the 20ZES1-pACO strain (Fig. 5).
Furthermore, improving the ornithine pool (i.e., the direct precursor of putrescine) would be necessary to improve the putrescine titer. Ornithine was synthesized from glutamate by five sequential catalytic reactions catalyzed by N-acetylglutamate synthase (encoded by argA), acetylglutamate kinase (argB), N-acetylglutamate semialdehyde dehydrogenase (argC), and N-acetylornithine transaminase (argD) to yield N-acetylornithine. Ornithine was formed in a recycling pathway by bifunctional ornithine acetyltransferase (argJ), which catalyzed N-acetylglutamate from glutamate, and ornithine by transacetylation between N-acetylornithine and glutamate. M. alcaliphilum 20Z possesses the recycling pathway, which was recognized as an efficiency pathway for production of an ornithine-derived product such as putrescine; the recycling pathway was an efficient pathway compared to the linear pathway catalyzed by acetylornithine deacetylase (argE) found in E. coli, type II methanotrophs, and other species, which converted acetylornithine to ornithine and generated acetate as an intermediate product [28]. These genes are located in five different loci. Plasmid-based overexpression of argCJBDFRGH or argJ alone in C. crenatum SYPA 5-5 also led to enhanced arginine production, the downstream product of ornithine [29]. Overexpression of argJ increases flux from glutamate toward an ornithine biosynthesis pathway and efficient conversion of acetylornithine to ornithine.
Protein–protein association networks with speC were also investigated [30]. The interaction of argD with speC was found in a characterized methylotrophic strain, Methylobacterium extorquens AM1 [31]. Thus, argD and argJ, two native genes in M. alcaliphilum 20Z, were selected as targets for overexpression to improve the ornithine pool. N-acetylornithine transaminase (argD) and ornithine acetyltransferase (argJ) were cloned into pAWP89 under the control of a pTac promoter. Two flanking regions in the genome of M. alcaliphilum 20Z were constructed into vector pCM351 along with the pTac-argDJ fragment. Flank F2 was inserted into the SacI site, resulting in vector pCM351-F2. Flank F1 and pTac-argDJ fragments were inserted in the EcoRI and NotI sites to generate vector pCAR2. Vector pCAR2 was introduced into 20ZE3, and a double crossover allelic exchanged mutant 20ZE4A with the genotype ΔldhΔackΔspeE1::argDJ was successfully obtained. In these strains, putrescine production was improved by 21.08% and 51.57% compared to 20ZE3-pACO and 20ZES1-pACO, respectively. A maximized titer of 39.04 ± 1.35 mg/L (approximately three times higher than 20Z-pACO) was obtained (Fig. 5).
Enhanced production of putrescine with nitrogen source optimization
Although ammonia was considered to be the best inorganic nitrogen source for E. coli because it provided the fastest growth rate [32], it is not widely used for culturing methanotrophs. Currently, nitrate is the main nitrogen source for culturing methanotrophs. Nitrate is subsequently reduced to nitrite and finally to ammonia by nitrate reductase and nitrite reductase [33]. Ammonia was then assimilated by glutamate dehydrogenase (GDH) or the glutamine synthetase/glutamate synthase (GS/GOGAT) pathway. Nitrate assimilation is an energetically expensive process, which is not favorable for growth of bacteria and for glutamate-derived products. In addition, the GDH pathway was stimulated in the presence of ammonia, leading to direct assimilation of ammonia to glutamate. Therefore, we investigated the effect of different nitrogen sources on the growth of M. alcaliphilum 20Z and putrescine production from methane.
Wild-type and the engineered strains 20ZE4A-pACO were grown in nitrate mineral salt medium (NMS) with 10 mM of potassium nitrate and in an ammonium mineral salt medium (AMS) with various concentrations of ammonium chloride: 1 mM, 2 mM, 5 mM, 10 mM, and 20 mM. Unfortunately, no growth of M. alcaliphilum 20Z was observed in AMS media at any concentration of ammonium chloride. Due to the lack of substrate specificity, methane monooxygenases oxidized ammonia to hydroxylamine, which led to an incompatibility between methane oxidation and ammonia oxidation. In addition, ammonia has potential toxicity to microorganism under high pH condition [34]. Cultivation of wild-type and engineered strains in lower pH was also conducted. The wild-type and engineered strains were slowly grown in NMS at neutral pH, but they did not grow in AMS at any pH (data not shown). Therefore, ammonia toxicity is mainly caused by the incompatibility of ammonia oxidation and methane oxidation and toxicity of hydroxylamine—an ammonia oxidation product [35, 36]. Ammonia is not a suitable nitrogen source for growth in methane. However, an appropriate amount of ammonium chloride supplement during growth in NMS can enhance putrescine production. Growth of M. alcaliphilum 20Z in ammonia nitrate mineral salt medium (ANMS) with various concentrations of ammonium chloride was also examined.
The performance of wild-type and engineered strains 20ZE4A-pACO in ANMS with various concentrations of ammonium chloride (1 mM, 2 mM, 5 mM of NH4Cl) was examined. With the supplement of 1 mM ammonium chloride, the wild-type and the engineered strains 20ZE4A-pACO showed higher growth rate in comparison with growth in NMS. With the supplement of 2 mM ammonium chloride, the growth of wild-type and engineered strains showed longer lag phase in the first 48 h of growth. However, after 96 h, the engineered strains reached 1.12-fold higher optical density than wild type, and the engineered strain reached highest optical density (OD600 = 4.0) compared to that of wild type (OD600 = 2.5) in flask culture (Fig. 6a). However, no growth of either wild-type or the engineered strain was observed with 5 mM of ammonium chloride.
After 268 h of cultivation, the engineered strain 20ZE4A-pACO produced 39.14 ± 1.35 mg/L of putrescine in an NMS medium. On the other hand, this engineered strain was able to produce 59.46 ± 0.92 and 98.08 ± 2.86 mg/L of putrescine in ANMS with 1 mM and 2 mM of ammonium chloride, respectively (Fig. 6b). In ANMS with 2 mM of ammonium chloride, the highest titer of 98.08 mg/L putrescine with a productivity of 2.9 nmol/gDCW/h and a yield of 0.0276 g-(putrescine)/g-CH4 was obtained. This was approximately 2.5 times higher than that obtained in NMS and is the highest titer of putrescine production from methane.
Analysis of gene expression level in response to change of nitrogen sources
For further understanding how gene expression is regulated in response to the change of nitrogen sources, transcriptome shotgun sequencing (RNA-seq) of M. alcaliphilum 20Z strain 20ZE4A-pACO cultured in ANMS and NMS was analyzed. Different expression levels of genes involved in ammonia assimilation, TCA cycle (Fig. 7a), and central metabolic pathway (Fig. 7b) were estimated as logarithm base twofold change (log2FoldChange) of gene expression level in NMS versus ANMS. Log2FoldChange < 0 of the given gene means upregulation of this gene in ANMS compared to NMS.
Among genes involved in ammonia assimilation in ANMS, only GOGAT was upregulated, while both GDH and GS were downregulated. Genes involved in ornithine biosynthesis pathway, argC, argD, and argJ, were highly upregulated in ANMS, especially, argJ was strongly upregulated with five-fold changes. Genes of the upstream of α-ketoglutarate dehydrogenase in TCA cycle were highly upregulated in ANMS, and only slight changes were observed in the downstream genes with some downregulation. Genes in the EMP pathway and methane assimilation were mostly upregulated in ANMS. Interestingly, methanol dehydrogenase large subunit mxaF and a downstream gene mxaJ were 19-fold upregulated in ANMS compared to NMS. Genes in methane assimilation, upstream reactions of TCA cycle, ammonia assimilation (GOGAT)n and ornithine biosynthesis pathway were significantly upregulated in ANMS. It can be inferred that small amount of ammonia, which reduced the incompatible inhibition of methane oxidation and ammonia oxidation, could be directly assimilated into glutamate. The saved energy consumption due to the decrease of nitrate assimilation might enhance the methane assimilation, together with 19-fold upregulation of methanol dehydrogenase large subunit in ANMS compared to NMS. As a result, a high titer of putrescine along with high growth rate and cell density was obtained in the ANMS medium.