Evaluation of Heterologous Biosynthetic Pathways for Methanol-Based 5-Aminovalerate Production by Thermophilic Bacillus methanolicus

The use of methanol as carbon source for biotechnological processes has recently attracted great interest due to its relatively low price, high abundance, high purity, and the fact that it is a non-food raw material. In this study, methanol-based production of 5-aminovalerate (5AVA) was established using recombinant Bacillus methanolicus strains. 5AVA is a building block of polyamides and a candidate to become the C5 platform chemical for the production of, among others, δ-valerolactam, 5-hydroxy-valerate, glutarate, and 1,5-pentanediol. In this study, we test five different 5AVA biosynthesis pathways, whereof two directly convert L-lysine to 5AVA and three use cadaverine as an intermediate. The conversion of L-lysine to 5AVA employs lysine 2-monooxygenase (DavB) and 5-aminovaleramidase (DavA), encoded by the well-known Pseudomonas putida cluster davBA, among others, or lysine α-oxidase (RaiP) in the presence of hydrogen peroxide. Cadaverine is converted either to γ-glutamine-cadaverine by glutamine synthetase (SpuI) or to 5-aminopentanal through activity of putrescine oxidase (Puo) or putrescine transaminase (PatA). Our efforts resulted in proof-of-concept 5AVA production from methanol at 50°C, enabled by two pathways out of the five tested with the highest titer of 0.02 g l–1. To our knowledge, this is the first report of 5AVA production from methanol in methylotrophic bacteria, and the recombinant strains and knowledge generated should represent a valuable basis for further improved 5AVA production from methanol.


INTRODUCTION
The worldwide amino acid market is progressively growing at 5.6% annual rate and is estimated to reach US$25.6 billion by 2022, with amino acids used for animal feed production being its largest component (Wendisch, 2020). The growing demand for amino acid supply confronts the biotechnological industry with an unprecedented challenge of identifying suitable feedstocks, especially in terms of replacing sugars and agricultural products, use whereof deteriorates food supply and threatens biodiversity (Cotton et al., 2020). Methanol, together with other one-carbon (C1) compounds, is considered a very promising substitute for feedstock that are conventionally used in biotechnological processes. The major advantages of using methanol as carbon source are its low production cost (e.g., methanol from steam reforming of methane), ease of transport and storage, and complete miscibility that bypasses the mass transfer barrier and potentially supports improvement in microbial productivities. However, what seems to cause a considerable difficulty in propagation of methanol as biotechnological feedstock is the limited selection of microorganisms capable to be used as their carbon and energy source. One of the compelling candidates to become a workhorse for the methanol-based production of amino acids is Bacillus methanolicus, a thermophilic methylotroph isolated from freshwater marsh soil by Schendel et al. (1990). The wild-type strain MGA3 naturally overproduces L-glutamate in methanol-controlled fed-batch fermentations with volumetric titers reaching up to 60 g l −1 (Heggeset et al., 2012; Table 1). Furthermore, thanks to recent developments in the toolbox for gene overexpression, it was engineered for production of different amino acid derivatives such as γaminobutyric acid and cadaverine (Naerdal et al., 2015;Irla et al., 2017; Table 1). MGA3 produces 0.4 g l −1 of Llysine in high cell density fed-batch fermentations (Brautaset et al., 2010; Table 1); this titer was improved nearly 30fold up to 11 g l −1 by plasmid-based overexpression of a gene coding for aspartokinase, a key enzyme controlling the synthesis of aspartate-derived amino acids (Jakobsen et al., 2009). Through application of a classical mutagenesis technique, a derivative of B. methanolicus MGA3 (M168-20) was constructed, which produces 11 g l −1 of L-lysine in high cell density methanol-controlled fed-batch fermentations (Brautaset et al., 2010); the L-lysine overproduction being caused among others by mutation in the hom-1 gene coding for homoserine dehydrogenase (Hom) and in the putative lysine 2,3-aminomutase gene (locus tag BMMGA3_02505). The mutation in hom-1 leads to the loss of catalytic activity of homoserine dehydrogenase and redirection of metabolic flux toward the L-lysine pathway and therefore its accumulation (Naerdal et al., 2011. 5-Aminovalerate (5AVA) is a product of L-lysine degradation, and it is mainly synthesized in a two-step process catalyzed by a lysine monooxygenase (DavB) and a δ-aminovaleramide amidohydrolase (DavA) (Revelles et al., 2005). 5AVA is a nonproteogenic five-carbon amino acid that could potentially be used as building block for producing biobased polyamides (Adkins et al., 2013;Park et al., 2014;Wendisch et al., 2018). It is also a promising precursor for plasticizers and chemicals that are intermediates for bioplastic preparation: δ-valerolactam (Chae et al., 2017), 5-hydroxy-valerate (Sohn et al., 2021), glutarate (Adkins et al., 2013;Pérez-García et al., 2018), and 1,5-pentanediol (Cen et al., 2021). As summarized in Table 1, diverse approaches have been made at the establishment of microbial 5AVA production. Pseudomonas putida KT2440, which possesses davBA in its genome, can synthesize 20.8 g l −1 5AVA from 30 g l −1 L-lysine in 12 h (Liu et al., 2014). Production of 5AVA was established in Corynebacterium glutamicum by heterologous overexpression of the DavB-and DavA-encoding genes (davBA) from P. putida with a final titer up to 39.9 g l −1 in a sugar-based fed-batch fermentation (Rohles et al., 2016;Shin et al., 2016;Joo et al., 2017). 5AVA can be also produced in a process of bioconversion of L-lysine supplemented to the growth medium with molar yields of up to 0.942 achieved by Escherichia coli strains overproducing DavBA (Park et al., 2014;Wang et al., 2016). Moreover, when the recombinant E. coli strain expressing davAB genes was cultured in a medium containing 20 g l −1 glucose and 10 g l −1 L-lysine, 3.6 g l −1 5AVA was produced, representing a molar yield of 0.45 (Park et al., 2013). Disruption of native lysine decarboxylase (CadA and LdcC) activity in E. coli strains overexpressing davBA limited cadaverine by-product formation, enabling increased accumulation of Llysine following 5AVA production, with 5AVA yield of 0.86 g l −1 in glucose-based shaking flask fermentation (Adkins et al., 2013). Furthermore, Cheng et al. (2018) reported that the oxidative decarboxylation of L-lysine catalyzed by a L-lysine α-oxidase (RaiP) from Scomber japonicus led to 5AVA production. The production of RaiP was enhanced by the addition of 4% (v/v) ethanol and 10 mM H 2 O 2 , which increased the 5AVA titer to 29.12 g l −1 by an E. coli host strain in a fed-batch fermentation (Cheng et al., 2018). Recently, in a similar L-lysine bioconversion strategy, an E. coli whole-cell catalyst producing RaiP was developed, converting 100 g l −1 of L-lysine hydrochloride to 50.62 g l −1 5AVA representing a molar yield of 0.84 (Cheng et al., 2020).
Recent efforts have employed novel metabolic routes toward 5AVA. In Pseudomonas aeruginosa PAO1, the set of enzymes composed of glutamylpolyamine synthetase, polyamine:pyruvate transaminase, aldehyde dehydrogenase, and glutamine amidotransferase is essential for the degradation of diamines through the γ-glutamylation pathway (Yao et al., 2011), which may lead to 5AVA production when cadaverine is degraded (Luengo and Olivera, 2020). Jorge et al. (2017) established a three-step 5AVA biosynthesis pathway consisting of the conversion of L-lysine to cadaverine by the activity of the enzyme LdcC, followed by cadaverine conversion to 5AVA through consecutive transamination, by a putrescine transaminase (PatA), and oxidation by a PatD. The heterologous overexpression of the genes ldcC, patA, and patD led to 5AVA production to a final titer of 5.1 g l −1 by an engineered C. glutamicum strain in a shake flask fermentation (Jorge et al., 2017). This pathway has served as basis for the establishment of a new three-step pathway toward 5AVA using the monooxygenase putrescine oxidase (Puo), which catalyzes the oxidative deamination of cadaverine, instead of PatA .
Critical factors that can affect 5AVA accumulation in a production host are the presence of a native 5AVA degradation pathway in its genome and the end product-related inhibition. In some bacterial species, such as P. putida KT2440, Pseudomonas syringae, Pseudomonas stutzeri, and C. glutamicum, 5AVA is degraded by a GABAse (Figure 1), composed of two enzymes γ-aminobutyric acid aminotransferase (GabT) and succinic 1 | Comparison of the 5AVA production by different engineered microbial strains and production of amino acids by B. methanolicus.

Organism
Approach 5AVA titer [g l −1 ] References Pseudomonas putida KT2440 DavBA-based biocatalytic production of 5AVA from 30 g l −1 L-lysine 20.80 Liu et al., 2014 Corynebacterium glutamicum Heterologous expression of davBA; sugar-based fed-batch fermentation 33. 10 Shin et al., 2016 28.00 Rohles et al., 201639.93 Joo et al., 2017 Heterologous expression of ldcC and patAD; shake flask fermentation 5. 10 Jorge et al., 2017 Heterologous expression of puo and patD, deletion of gabTD; microbioreactor fermentation 3.70 Haupka et al., 2020 Escherichia coli Heterologous expression of davBA and deletion of cadA; glucose-based shaking flasks fermentation 0.86 Adkins et al., 2013 Heterologous expression of davBA; sugar-based fermentation; 10 g l −1 lysine provided 3.60 Park et al., 2013 Heterologous expression of davBA; sugar-based fed-batch fermentation 0.50 Park et al., 2013 Heterologous expression of davBA; glucose-based fed-batch fermentation; 120 g l −1 L-lysine provided 90.59 Park et al., 2014 Heterologous expression of davBA; fed-batch whole-cell bioconversion of L-lysine maintained at 120 g l −1 semialdehyde dehydrogenase (GabD) (Park et al., 2013;Rohles et al., 2016;Pérez-García et al., 2018); for example, GABAse from Pseudomonas fluorescens KCCM 12537 retains 47.7% activity when 5AVA is used as its substrate in comparison to when GABA is used (So et al., 2013). Based on the previous research, B. methanolicus seems a feasible candidate for 5AVA production because it does not possess the necessary genetic background for GABAse-based 5AVA degradation, lacking the gabT gene in its genome . It was reported that 5AVA does not supports growth of B. methanolicus neither as sole carbon source nor as sole nitrogen source (Haupka et al., 2021). However, B. methanolicus displays low tolerance to 5AVA, with growth being impaired by addition of 1.17 g l −1 5AVA to the culture broth (Haupka et al., 2021). Even though the application of diverse 5AVA biosynthetic pathways has led to significant improvement in titers and yields of 5AVA production in bacterial hosts, the most efficient processes rely on raw materials that contain sugar and/or agricultural products. Addressing shortages of global resources and food requires a replacement of the current mode of industrial biotechnology, which results in the need for novel biosynthetic pathways that utilize alternative raw materials such as methanol. Hence, in the present study we have selected five different pathways to establish methanol-based 5AVA production in the methylotrophic bacterium B. methanolicus. For two of the five pathways, proof-of-principle 5AVA production was achieved and our results should represent a valuable basis of knowledge and strains for further improved 5AVA production from methanol at 50 • C.
AB937978.1) and S. japonicus (GenBank AB970726.1) were codon-optimized for B. methanolicus MGA3 expression and synthesized by Twist Biosciences (Supplementary Table S1 and Supplementary Material). The davBA operons from alternative hosts Williamsia sterculiae CPCC 203464, Roseobacter denitrificans OCh 114 strain DSM 7001, and Parageobacillus caldoxylosilyticus B4119 (davA only) were codon-optimized for expression in B. methanolicus, synthesized and provided in the pUC57 plasmid from GenScript (Supplementary Table S1 and Supplementary Material). Isolated genomic DNA of Bacillus megaterium DSM32 was purchased from German Collection of Microorganisms and Cell Cultures GmbH (DSMZ). All primers (Sigma-Aldrich) used in this research are listed in Table 2.

Molecular Cloning
The E. coli DH5α competent cells were prepared according to the calcium chloride protocol as described in Green and Rogers (2013) or purchased as chemically competent NEB 5-α E. coli cells (New England Biolabs). All standard molecular cloning procedures were carried out as described in Sambrook and Russell (2001) or according to manuals provided by producers. Chromosomal DNA was isolated as described in Eikmanns et al. (1994). PCR products were amplified using CloneAmp HiFi PCR Premix (Takara) and purified using a QIAquick PCR Purification Kit from Qiagen. DNA fragments were separated using 8 g l −1 SeaKem LE Agarose gels (Lonza) and isolated using a QIAquick Gel Extraction Kit (Qiagen). The colony PCR was performed using GoTaq DNA Polymerase (Promega). The sequences of cloned DNA fragments were confirmed by Sanger sequencing (Eurofins). B. methanolicus MGA3 was made electrocompetent and transformed by electroporation as described previously (Jakobsen et al., 2006). Recombinant DNA was assembled in vitro by means of the isothermal DNA assembly method (Gibson et al., 2009), employing the NEBuilder HiFi DNA Assembly Kit or ligation with T4 DNA ligase.  pMI2mp plasmid was obtained via site-directed mutagenesis (SDM) of pTH1mp performed as previously described with CloneAmp HiFi PCR Premix (Liu and Naismith, 2008). The detailed description of plasmid creation is presented in Supplementary Material.

Determination of Amino Acid Concentration
For the analysis of amino acid concentrations, 1 ml of the culture sample was taken from the bacterial cultures and centrifuged for 10 min at 11,000 rpm. Extracellular amino acids were quantified by means of high-pressure liquid chromatography (HPLC, Waters Alliance e2695 Separations Module). The samples underwent FMOC-Cl (fluorenylmethyloxycarbonyl chloride) derivatization before the analysis, according to the protocol described before (Haas et al., 2014), and were separated on a column (Symmetry C18 Column, 100 Å, 3.5 µm, 4.6 mm × 75 mm, Waters) according to the gradient flow presented in Table 3, where A is an elution buffer 50 mM Na-acetate pH = 4.2 and B is an organic solvent, acetonitrile.
The detection was performed with a Waters 2475 HPLC Multi Fluorescence Detector (Waters), with excitation at 265 nm and emission at 315 nm.

Enzyme Assays
In order to determine enzymatic activity, crude extracts of recombinant B. methanolicus cells were prepared according to Drejer et al. (2020). B. methanolicus strains were inoculated in SOB medium and grown to exponential phase (OD 600 = 0.8).
Recombinant expression was induced by addition of 10 g l −1 xylose 2 h after inoculation. A total amount of 50 ml culture broth was harvested by centrifugation at 7,500 rpm and 4 • C for 15 min and washed twice in ice-cold buffer used for specific enzyme assay before storing at −80 • C. The cells were thawed in ice and disrupted by sonication using a Fisherbrand Sonic Dismembrator (FB-505) with 40% amplitude with 2 s on and 1 s off-pulse cycles for 7 min. Cell debris was then removed by centrifugation (at 14, 000 rpm and 4 • C for 1 h). Protein concentrations were determined by Bradford assay (Bradford, 1976), using bovine albumin serum (Sigma) as standard. L-Lysine α-oxidase activity was assayed by measuring the rate of hydrogen peroxide formation, as described elsewhere (Tani et al., 2015a). The reaction was initiated by adding crude extracts from B. methanolicus strains to the reaction media (50 • C) consisting of 100 mM L-lysine and 50 mM pH 7 phosphate buffer, resulting in a total volume of 1 ml. Next, the sample was quenched by addition of 50 µl 2 M HCl. After neutralization with 50 µl 2 M NaOH, 200 µl of the mixture was withdrawn and transferred to 800 µl of a second reaction mixture containing 50 mM pH 6 phosphate buffer, 30 mM phenol, 2 units ml −1 peroxidase from horseradish (Sigma) and 0.5 mM 4aminoantipyrine. Formation of quinoneimine dye from oxidative coupling of phenol and 4-aminoantipyrine (Job et al., 2002) was determined by measuring absorbance at 505 nm using a Cary 100 Bio UV-visible spectrophotometer (Varian). One unit (U) of RaiP activity was defined as the amount of enzyme that catalyzes the formation of 1 µmol hydrogen peroxide per minute.
Catalytic activities of PatA and PatD or putrescine oxidase and PatD were measured by using a coupled reaction, and cadaverine was used as substrate instead of putrescine, as previously described elsewhere, with modifications (Jorge et al., 2017). The 1-ml assay mix contained 0.1 M Tris-HCl pH 8.0, 1.5 mM α-ketoglutarate, 2.5 mM cadaverine, 0.1 mM pyridoxal-5 -phosphate, and 0.3 mM NAD. In this coupled reaction, cadaverine was converted to 5AVA via 5-aminopentanal and one unit of coupled enzyme activity was defined as the amount of the enzyme that formed 1 µmol of NADH (ε340 nm = 6.22 mM −1 cm −1 ) per minute at 50 • C.
The coupled DavAB assay was performed as described in Liu et al. (2014) with some modifications. Five hundred microliters of crude extract was added into 50-ml Falcon tubes filled with 4 ml 100 mM phosphate buffer pH 7.0 supplemented with 10 g l −1 L-lysine. The tubes were incubated for 40 h at 30 or 50 • C with stirring at 200 rpm. The samples for quantification of 5AVA concentration through HPLC (see section "Determination of Amino Acid Concentration") were taken at the beginning of incubation, after 16 h and after 40 h.

Selection, Design, and Construction of Heterologous Biosynthetic Pathways for 5AVA Biosynthesis in B. methanolicus
Due to the fact that B. methanolicus is a thermophile, a typical issue concerning implementation of biosynthetic pathways from heterologous hosts is the lack of thermostability of the transferred enzymes. It was shown before that a screening of diverse donor organisms allows to identify pathways active at 50 • C and leads to increased product titers Drejer et al., 2020). In order to extend the scope of our screening, we have constructed 26 strains with five different 5AVA biosynthetic pathways, which are presented in Figure 1, derived from diverse donors. Two pathways that directly convert L-lysine to 5AVA were chosen: the DavBA pathway ( Figure 1A) and the RaiP pathway ( Figure 1B), as well as three pathways that use cadaverine as an intermediate: the SpuI pathway (Figure 1C), the PatA pathway (Figure 1D), and the Puo pathway ( Figure 1E).
The genes encoding the core part of those pathways are cloned into a θ-replication, low copy number derivative of pHCMC04 plasmid, pBV2xp, under control of a B. megateriumderived, xylose-inducible promoter, and the genes encoding any ancillary enzymes are cloned into pTH1mp or pMI2mp plasmids, which are compatible to pBV2xp, under control of the mdh promoter (Irla et al., 2016). The plasmids with genes encoding desired pathways were constructed as described fully in the Supplementary Material and then used to transform B. methanolicus cells leading to formation of strains presented in Table 4.
With help of retrosynthesis analysis, we have considered two pathways that utilize L-lysine directly as precursor and that utilize either DavB (EC 1.13.12.2) and DavA (EC 3.5.1.30) activity (DavBA pathway, Figure 1A) or RaiP (EC 1.4.3.14) in the presence of H 2 O 2 (RaiP pathway, Figure 1B) for further conversion into 5AVA. For DavBA production, three different davBA operons from the following mesophilic organisms were applied: P. putida, W. sterculiae, and R. denitrificans. We could not identify a complete davBA operon from a thermophilic host; however, thermophilic P. caldoxylosilyticus possesses a putative davA gene and was also included in this study. All selected davBA operons were codon-optimized and cloned into the pBV2xp vector under control of the xylose-inducible promoter as described in the (Table 4) carried heterologous raiP gene sequences from the prokaryote P. simplex and from the eukaryotic genetic donors S. japonicus and T. viride, respectively, the two latter with characterized RaiP activity (Arinbasarova et al., 2012;Tani et al., 2015a). The full length of codon-optimized sequences derived from S. japonicus and T. viride is present in the Supplementary  Table S1. The original S. japonicus sequence encodes a protein with 617 amino acids and has a 52.2% GC content, while the sequence codon optimized for B. methanolicus has a GC content of 29%. The T. viride-derivative sequence was adjusted from the GC content of 42.5 to 28.6%. The substitution of nucleotides did not alter their coding amino acid sequences.
Another pathway, also predicted by our retrosynthesis analysis, potentially leading to production of 5AVA from Llysine is a three-step pathway composed of CadA, PatA (EC 2.6.1.82, PatA), and 5-aminopentanal dehydrogenase (EC 1.2.1.19, PatD) (PatA pathway, Figure 1D). In order to test this pathway, two strains were constructed, MGA3_PatA Ec and MGA3_PatA Bm , through transformation of B. methanolicus with pTH1mp-cadA plasmid, and pBV2xp-AVA Ec or pBV2xp-AVA Bm , respectively ( Table 4). As described in the Supplementary Material, the lysine decarboxylase-encoding gene (cadA) was placed under control of the mdh promoter in a rolling circle vector pTH1mp. The E. coli-derived patAD operon encoding previously characterized enzymes was placed under control of the xylose-inducible promoter in pBV2xp, resulting in pBV2xp-AVA Ec (Samsonova et al., 2003). The genes of the patAD operon in B. megaterium were identified based on a BLAST search of its genome and were cloned into pBV2xp, yielding pBV2xp-AVA Bm (Altschul et al., 1990). While the existence of prior art makes it a solid candidate, we knew that its second step catalyzed by PatA may suffer from an unfavorable thermodynamic (predicted close to 0 kJ mol −1 ) (Noor et al., 2012).
In our study, we have also included a pathway confirmed through retrosynthesis analysis where the step of cadaverine transamination (PatA pathway, Figure 1D) is replaced by its oxidative deamination (Puo pathway, Figure 1E) because this reaction displays a more favorable thermodynamic (predicted close to −100 kl mol −1 in cell conditions) in comparison to PatA. While a cadaverine oxidase has not been identified before, it was shown that putrescine oxidase encoded by puo retains up to 14% of its maximal activity when cadaverine is used as a substrate (Okada et al., 1979;Ishizuka et al., 1993;van Hellemond et al., 2008;. We have therefore decided to express three different versions of the puo gene derived from K. rosea, P. aurescens, and R. qingshengii, together with the E. coli-derived patD gene from the pBV2xp plasmid (for details see Supplementary Material), which led to creation of the following strains: MGA3_Puo Kr , MGA3_Puo Pa , and MGA3_Puo Rq , respectively (Table 4). In order to prevent oxidative stress caused by H 2 O 2 formation, a native gene encoding catalase was homologously expressed from pTH1mp plasmid in all constructed strains.

Testing Recombinant B. methanolicus Strains for 5AVA Production From Methanol
The plasmids designed and built as described in the above Section were used for transformation of wild-type B. methanolicus cells and resulted in the creation of 26 different strains (Table 4) which were then tested for their ability to synthetize 5AVA. All strains were cultivated in minimal medium supplemented with methanol as the sole carbon and energy source, and the 5AVA titer was evaluated after the strains had reached the stationary growth phase as described in the following sections.

Expression of the DavAB-Encoding Genes Resulted in no 5AVA Biosynthesis in B. methanolicus
In the first attempt, we heterologously expressed genes encoding the DavBA pathway in B. methanolicus MGA3 (Figure 1A). In addition to the well-known davBA operon from P. putida (gamma-proteobacteria), the alternative davBA operon from W. sterculiae (actinobacteria) and davAB from R. denitrificans (alpha-proteobacteria) were tested for 5AVA formation in B. methanolicus MGA3. Moreover, the only enzyme identified from a thermophilic host, DavA from P. caldoxylosilyticus (bacilli), was combined with the before mentioned lysine 2-monooxygenases (DavB). P. caldoxylosilyticus has a reported optimum growth temperature from 50 to 65 • C (Fortina et al., 2001).
Several considerations were made with regard to strain design, namely, adjusting the GC content and the types of codons present in the open reading frames in the genomic DNA of a donor and designing suitable expression cassettes. In total, seven different B. methanolicus strains were constructed: Table 4). However, in none of the tested strains (MGA3_DavBA Pp , MGA3_DavBA Ws , MGA3_DavBA Rd , MGA3_DavB Ws A Pc , MGA3_DavA Pc B Rd ), the active pathway was expressed; and followingly no 5AVA accumulation was observed during shake flask cultivations in any constructed strain (data not shown).
The first reaction step from L-lysine to 5-aminopentanamide requires O 2 (Figure 1A), and due to the high O 2 demand to facilitate the assimilation of methanol, we also tested 5AVA formation from the alternative carbon source mannitol. Neither was this strategy successful. Furthermore, the DavAB pathway was also tested in the genetic background of L-lysineoverproducing B. methanolicus strain M160-20. Specifically, the following strains were constructed: M168-20_DavBA Pp , M168-20_DavA Pp B Pp (2p), and M168-20_DavA Pp B Ws (2p); however, none of them produced any detectable 5AVA (data not shown). Taken together, the DavBA pathway did not enable 5AVA formation. It is not clear whether this was caused by low enzymatic stability at 50 • C (only P. caldoxylosilyticus is known to be thermophilic among the organisms found to be source organisms for the two genes). In order to exclude the effect of elevated temperature on the DavAB activity, we tested enzymatic activity at 30 • C for selected strains (MGA3_DavBA Pp , MGA3_DavBA Ws , MGA3_DavBA Rd , MGA3_DavB Ws A Pc , and MGA3_DavA Pc B Rd ); however, no DavAB activity was detected (data not shown). The reason why the functional DavAB pathway was not expressed in B. methanolicus remains unknown.

RaiP Pathway Is Functional in B. methanolicus and Supports 5AVA Production
Methanol-based 5AVA biosynthesis was attempted via heterologous expression of RaiP encoding gene raiP in MGA3. The strains MGA3(pBV2xp-raiP Ps ) named MGA3_RaiP Ps , MGA3(pBV2xp-raiP Sj ) named MGA3_RaiP Sj , and MGA3(pBV2xp-raiP Tv ) named MGA3_RaiP Tv (Table 4) carry the raiP gene from the bacterium P. simplex and raiP genes with codon-optimized sequences from the eukaryotic donors S. japonicus and T. viride, respectively. The T. viridederived RaiP was shown to be stable at temperatures up to 50 • C (Arinbasarova et al., 2012). It is reported that the RaiP protein from S. japonicus is thermally stable for at least 1 h in temperatures up to 60 • C, with its highest activity registered at 70 • C (Tani et al., 2015b). Moreover, although there is no kinetic characterization of RaiP from P. simplex available, this bacterium is classified as mesophilic, with growth optimum at 30 • C (Yumoto et al., 2004). To examine the activity of RaiP in the constructed B. methanolicus strains, L-lysine α-oxidase activity was measured at 50 • C. While the empty vector control strain has shown no RaiP activity, the highest RaiP specific activity was observed in crude extracts from strain MGA3_RaiP Tv , being 62.1 ± 1.4 mU mg −1 (Figure 2A). The values of RaiP activity for strains MGA3_RaiP Ps and MGA3_RaiP Sj were 1.4 ± 0.3 mU mg −1 and 12.0 ± 4.4 mU mg −1 , respectively (Figure 2A). It is not clear if the poor activity of heterologous RaiP from genetic donors S. japonicus and P. simplex was caused by low enzymatic stability at 50 • C, and the reason for that remains to be investigated.
HPLC analysis of supernatant from MGA3_RaiP Tv strain cultivated in minimal medium revealed 16.15 ± 1.62 mg L −1 5AVA and 0.27 ± 0.04 mg L −1 L-lysine. In contrast, the L-lysine level in the MGA3 strain harboring the empty vector plasmid pBV2xp (MGA3_EV) was 37.8 ± 7.2 mg L −1 (Figure 2B). Even though a slight RaiP activity was observed in crude extract of the strains MGA3_RaiP Ps and MGA3_RaiP Sj , no 5AVA production was observed for those strains (data not shown). Let us note here that the 5AVA titer in the methanol-based shaking flask fermentation of strain MGA3_RaiP Tv was significantly inferior to that in previously reported glucose-based fermentations in E. coli (Cheng et al., 2018).
The value of the Michaelis-Menten constant for T. viridederived RaiP for L-lysine has been estimated (K m = 5.85 mg L −1 ) (Kusakabe et al., 1980). Therefore, the precursor levels in the B. methanolicus strains should not be a limiting factor for production of 5AVA. The RaiP-mediated production is mainly utilized in the L-lysine bioconversion approach, utilizing E. coli strains as whole-cell biocatalysts (Cheng et al., 2018(Cheng et al., , 2020(Cheng et al., , 2021 where high concentrations of the precursor were used; for example, the molar yield of 0.942 was obtained from 120 g l −1 Llysine (Park et al., 2014). However, construction and testing of the B. methanolicus strains M168-20_RaiP Sj , M168-20_RaiP Ps , and M168-20_RaiP Tv (Table 4), based on the L-lysine-over producing mutant M168-20 (Brautaset et al., 2010), did not result in any improved 5AVA production (data not shown).
The lack of 5AVA production in MGA3_RaiP Ps and MGA3_RaiP Sj , as well as low 5AVA titer produced by strain MGA3_RaiP Tv , might be related to the spontaneous conversion step that follows RaiP activity. This could be a limiting factor for the RaiP-mediated production of 5AVA. Three compounds are produced in a reaction catalyzed by RaiP: α-ketolysine, NH 3 , and H 2 O 2 (Mai-Prochnow et al., 2008;Cheng et al., 2018). In a second spontaneous step of 5AVA synthesis, the intermediate α-ketolysine is oxidatively decarboxylated to form 5AVA in the presence of H 2 O 2 as an oxidizing agent. It was shown that the addition of H 2 O 2 into the culture broth has led to an 18−fold increase of 5AVA titers in comparison with the control condition without H 2 O 2 (final titer 29.12 g l −1 ) in a 5-l fermenter (Cheng et al., 2018). The RaiP-mediated 5AVA production may be increased by enzymatic conversion of αketolysine in an approach different to ours, where spontaneous reaction of oxidative decarboxylation occurs. Recently, an artificial synthetic pathway for the biosynthesis of 5AVA in E. coli was developed, consisting of three steps: conversion of L-lysine to α-ketolysine via RaiP, decarboxylation of α-ketolysine to produce 5-aminopentanal via α-ketoacid decarboxylase, and oxidation of 5-aminopentanal to 5AVA via aldehyde dehydrogenase. The expression of the artificial pathway resulted in a yield increase of 774% compared to the single gene pathway (Cheng et al., 2021). This approach is potentially a feasible strategy we have shown in our study that E. coli-derived PatD is active as a 5-aminopentanal dehydrogenase in B. methanolicus and participates in 5AVA biosynthesis (see Section "The PatA Pathway Supports 5AVA Accumulation in B. methanolicus).

Use of the SpuI Pathway Does Not Lead to 5AVA Production in B. methanolicus
Three different pathways that use cadaverine as an intermediate product have been tested for their feasibility for production of 5AVA in B. methanolicus. Cadaverine biosynthesis in B. methanolicus cells was enabled through the activity of lysine decarboxylase encoded by a heterologously expressed cadA FIGURE 2 | Evaluation of RaiP enzyme activity (A) and amino acids production (B) in recombinant B. methanolicus strains. B. methanolicus strains MGA3_EV, MGA3_RaiP Sj , MGA3_RaiP Ps , or MGA3_RaiP Tv were cultivated in a shaking flask culture. The grown cells were harvested, washed twice with 50 mM phosphate buffer (pH 7.0), and disrupted by sonication. After centrifugation, the crude extracts were directly used for the RaiP assay. MGA3_EV and MGA3_RaiP Tv were cultivated for 27 h, and supernatants were obtained by centrifugation for HPLC analysis. The error bars represent standard deviation of technical triplicates. (Naerdal et al., 2015). Cadaverine can be converted to 5AVA through activity of a multistep diamine catabolic pathway derived from P. aeruginosa PAOI (SpuI pathway, Figure 1C) (Yao et al., 2011). The MGA3(pTH1mp-cadA)(pBV2xp-AVA Pp ) strain called MGA3_SpuI (Table 4) did not accumulate any 5AVA during methanol-based growth in minimal medium, despite the accumulation of the precursor, cadaverine, at the level of 118.8 ± 5.1 mg l −1 similar to the empty vector control strain (130.0 ± 5.3 mg l −1 ) ( Table 5). The cadaverine titers of 130.0 ± 5.3 mg l −1 achieved by MGA3_Cad are higher than the L-lysine titer of 37.8 ± 7.2 mg L −1 achieved by MGA3_EV in this study ( Figure 2B). This is in accordance with previous findings of Naerdal et al. (2011Naerdal et al. ( , 2015 who attributed high cadaverine titers for production strain in relation to L-lysine titer in empty vector MGA3_Kat 0.32 ± 0.00 0 ± 0 3.1 ± 0.5 Supplemented (500 mg L −1 ) 0.0 ± 0.0 MGA3_Puo Ec 0.28 ± 0.01 0 ± 0 5.0 ± 0.7 Supplemented (500 mg L −1 ) 0.0 ± 0.0 MGA3_Puo Pa 0.29 ± 0.01 0 ± 0 4.9 ± 0.9 Supplemented (500 mg L −1 ) 0.0 ± 0.0 MGA3_Puo Rq 0.29 ± 0.00 0 ± 0 3.7 ± 0.2 Supplemented (500 mg L −1 ) 0.0 ± 0.0 The B. methanolicus strains expressing pathways that use cadaverine as an intermediate (SpuI, PatA, or Puo pathways) were cultivated for 24 h, and supernatants were obtained by centrifugation for HPLC analysis. Catalytic activities of PatA and PatD or Puo and PatD were measured by using a coupled reaction, and cadaverine was used as substrate (see Section "Enzyme Assays"). The standard deviation of technical triplicates is shown. NB: RaiP activity and 5AVA production for the RaiP pathway is shown Figure 2.
control strain to a metabolic pull which deregulated flux through the L-lysine biosynthesis pathway.
The PatA Pathway Supports 5AVA Accumulation in B. methanolicus In the next step, two versions of the PatA pathway ( Figure 1D) derived from either E. coli or B. megaterium were tested in strains MGA3(pTH1mp-cadA)(pBV2xp-AVA Ec ) named MGA3_PatA Ec and MGA3(pTH1mp-cadA)(pBV2xp-AVA Bm ) named MGA3 _PatA Bm (Table 4), respectively. The optimal temperature of PatA derived from E. coli is 60 • C, which means that it is a thermostable enzyme that should be active at 50 • C, which is a temperature used for the production experiment. PatA was shown to have a broad substrate range including cadaverine and, in lower extent, spermidine, but not ornithine (Samsonova et al., 2003). This property was used by Jorge et al. (2017) who have shown in their study that it is possible to use PatA and PatD derived from E. coli to establish conversion of cadaverine to 5AVA, confirming experimentally the broad substrate range of those two enzymes. The B. megaterium-derived PatA was characterized only superficially with regard to its substrate spectrum and not optimal temperature or thermostability (Slabu et al., 2016); however, its host organism is known to have a wide temperature range for growth up to 45 • C (Vary et al., 2007). The multiple-sequence alignment with E. coli-derived enzymes showed identity of 63 and 38% for PatA and PatD, respectively (Okada et al., 1979). Both E. coli and B. megaterium-derived versions of the pathway are functional in B. methanolicus, with the combined PatAD activity of 7 ± 4 mU and 170 ± 37 mU mg −1 ( Table 5). Final 5AVA titers of 23.7 ± 2.7 and 8.3 ± 4.1 mg L −1 (Table 5) were achieved, which is considerably lower than 5AVA titers of 0.9 g l −1 obtained by wild-type C. glutamicum strain transformed with plasmids for expression of ldcC (coding for lysine decarboxylase) and patDA (Jorge et al., 2017). For both producer strains, the concentration of unconverted cadaverine is similar: 1.7 ± 0.1 mg l −1 and 1.5 ± 0.2 mg l −1 for MGA3_PatA Ec and MGA3_PatA Bm , respectively ( Table 5). While K m for cadaverine has not been assessed, it has been shown to be 811 mg l −1 for putrescine for E. coli-derived PatA; assuming similar K m for cadaverine, it may explain why full conversion of cadaverine has not occurred (Samsonova et al., 2003). Due to relatively high K m for putrescine of PatA, we decided to test how supplementation with external cadaverine affects 5AVA accumulation. In fact, for both MGA3_PatA Ec and MGA3_PatA Bm , 5AVA titers increased to 31.8 ± 2.3 and 77.7 ± 5.5, respectively, when the growth medium was supplemented with 500 mg l −1 cadaverine ( Table 5). These results indicate that the enhancement of precursor supply is one potential target for subsequent metabolic engineering efforts to increase 5AVA titers. Another important consideration for activity of transaminase is availability of keto acid that acts as amino group acceptor. It was shown that E. coli and B. megaterium-derived PatA can use either pyruvate or 2-oxoglutarate as amino group acceptors (Slabu et al., 2016); the intracellular concentrations of those compounds in B. methanolicus MGA3 cells are 3.2 and 2.7 mM, respectively (Brautaset et al., 2003). Knowing that K m for 2-oxoglutarate for E. coli-derived PatA is 19.0 mM (Samsonova et al., 2003), recovery of the keto acids may be a limitation for 5AVA accumulation. This issue could be potentially solved by heterologous production of alanine dehydrogenase or Lglutamate oxidase which catalyzes reactions where pyruvate or 2-oxoglutarate is produced (Böhmer et al., 1989;Sakamoto et al., 1990;Slabu et al., 2016).
Use of the Puo Pathway Leads to 5AVA Production in B. methanolicus Lastly, a pathway that relies on an activity of the monooxygenase putrescine oxidase (Puo, EC 1.4.3.10) was tested ( Figure 1E). Puo catalyzes the oxidative deamination of cadaverine in lieu of cadaverine transamination catalyzed by PatA. It was shown that different putrescine oxidases can use cadaverine as their substrate with 9-14% of their maximal activity shown when putrescine is a substrate (Desa, 1972;Okada et al., 1979;van Hellemond et al., 2008;. Moreover, putrescine oxidases derived from K. rosea (Micrococcus rubens) and Rhodococcus are thermostable and optimal activity of P. aurescens-derived Puo is at 50 • C (Desa, 1972;van Hellemond et al., 2008;. The disadvantage of this pathway is that it requires O 2 , the supply of which may be difficult to control. Furthermore, due to formation of hydrogen peroxide in the reaction catalyzed by Puo, the oxidative stress may increase when this pathway is active. In order to avoid detrimental effect of hydrogen peroxide accumulation, catalase was overproduced in the recombinant strains containing the Puo pathway: MGA3(pTH1mp-katA)(pBV2xp-AVA Kr ) named MGA3_Puo Kr , MGA3(pTH1mp-katA)(pBV2xp AVA Pa ) named MGA3_Puo Pa , and MGA3(pTH1mp-katA)(pBV2xp-AVA Rq ) named MGA3_Puo Rq (Table 4). To achieve sufficient levels of the pathway precursor, cadaverine, we have decided not to rely on plasmid-based production of lysine decarboxylase and to add cadaverine to the growth medium, instead. The tested recombinant strains with the Puo pathway did not produce 5AVA, which is consistent with no Puo-PatD activity detected in crude extracts ( Table 5). The Puo pathway was shown to be active in C. glutamicum where titer of 0.1 ± 0.0-0.4 ± 0.0 g l −1 5AVA was achieved .

CONCLUSION
In the search for 5AVA production from the sustainable feedstock methanol, we have screened five pathways toward 5AVA biosynthesis in B. methanolicus. No 5AVA production was observed for DavBA, Puo, and SpuI pathways. However, the pathways relying on RaiP and PatA activities were functional in shake flask cultures of B. methanolicus, which led to 5AVA production from methanol for the first time, respectively, up to 16.15 ± 1.62 mg l −1 or 23.7 ± 2.7. RaiP and PatA pathways are targets for further optimizations which could increase the 5AVA titers in the constructed strains. For instance, the improvement of substrate utilization and H 2 O 2 availability or decomposition efficiency might contribute to the increase in the yield of 5AVA. Moreover, our study shows that the availability of supplemented cadaverine has high impact on 5AVA titer when the PatA pathway is employed. Another factor that needs to be considered is tolerance to 5AVA, which was shown to be low (Haupka et al., 2021). Recently, adaptative laboratory evolution experiments resulted in the selection of a mutant strain of B. methanolicus that displays tolerance to approximately 46 g l −1 5AVA (Haupka et al., 2021), which could be employed as a platform to develop high-titer 5AVA production strains. This shows that methanol has the potential to become a sustainable feedstock for the production of 5AVA.

DATA AVAILABILITY STATEMENT
The original contributions generated for this study are included in the article/Supplementary Material, further inquiries can be directed to the corresponding author.