Unusual Aldehyde Reductase Activity for Production of Full-length Fatty Alcohol by Cyanobacterial Aldehyde Deformylating Oxygenase

Background: Aldehyde-deformylating oxygenase (ADO) is a non-heme di-iron enzyme that catalyzes deformylation of aldehydes to generate alkanes/alkenes. In this study, we report for the rst time that under anaerobic or limited oxygen conditions, Prochlorococcus marinus (PmADO) can generate full-length fatty alcohols from fatty aldehydes without eliminating a carbon unit. Results: Unlike the native activity of ADO which requires electrons from the Fd/FNR electron transfer complex, the aldehyde reduction activity of ADO requires only NADPH. Our results demonstrated that yield of alcohol products can be affected by oxygen concentration and type of aldehyde. Under O 2 -scant conditions (10-15%), yields of octanol and dodecanol were around 40-60% and could be increased up to 80% under strict anaerobic conditions (>0.0004%). Unexpectedly, Fe 2+ cofactor is not involved in the aldehyde reductase activity of PmADO because yields of alcohols obtained from holo- and apo-enzymes were similar under anaerobic conditions. The direct hydride transfer activity of PmADO is highly specic to substrates; NADPH not NADH can be used as a reductant to reduce medium-chain fatty aldehydes (C 6 C 10 ) with decanal as the most preferred substrate (the highest k cat /K m value with 98% bioconversion yield). Molecular dynamics (MD) simulations was used to identify a binding site of NADPH which is located close to the aldehyde binding site. In the metabolic engineered cells containing PmADO, dual activities of alkane and alcohol production could be detected. Conclusion: ndings which may be applied a


Background
Long-chain alcohols or fatty alcohols are molecules with amphiphilic property.The compounds and their derivatives are valuable and widely used in various industries as commodity chemicals such as detergents, surfactants, additives, personal care products, etc [1][2][3][4][5].The global production of fatty alcohol was above three million tonnes in 2015 [5].Currently, fatty alcohols can be produced from petroleumbased resources via ethylene polymerization and oxidation [6] or from bio-based materials such as triglyceride from palm oil industry via transesteri cation and hydrogenation [7,8] .Current methods for production fatty alcohols from both petro-based and bio-based materials are chemical processes which are not green and lead to high amount of CO 2 emission [9].Currently, industrial scale production of many valuable compounds including bio-alcohols from renewable feedstocks are possible due to advancement in synthetic biology, metabolic engineering and bioprocess [10][11][12].New and e cient biocatalysts for fatty alcohol production will contribute towards development of their production via bioprocess from renewable feedstocks which will help transform industries towards UN's sustainable development goals [9,10].
Several metabolic pathways and various types of microorganisms have been engineered for production of fatty alcohols [1,2,[13][14][15].However, the limitation lies at the production level of fatty alcohols which are required to increase titers, yields, productivities, and robustness in large-scale industrial process, making microbial production more cost-effective and commercially viable.Most of these engineered pathways rely on one-step or two-step reduction processes.The one-step reduction system uses the enzyme fatty acid reductase (FAR) to reduce fatty acyl ACP/CoA directly to yield fatty alcohols.The twostep reduction system uses two enzymes in which the rst step is reduction of fatty acyl ACP/CoA by fatty acyl ACP/CoA reductase (AAR/ACR) or reduction of free fatty acid by carboxylic acid reductase (CAR) to yield fatty aldehyde [16][17][18][19][20].The resulting fatty aldehyde is further converted to fatty alcohol by aldehyde reductase.However, high activities were observed for selected alcohols such as 1-propanol, 1butanol, 1-pentanol, isoamyl, 1-hexanol, 1-octanol, and benzyl alcohols with relatively high K m values [21].
Alternatively, if the engineered microbe contains a gene encoding an aldehyde-deformylating oxygenase (ADO), end product of the pathway is alkane/alkene instead of fatty alcohol [22,23].It has previously been reported that ADO can also convert fatty aldehydes to fatty alcohols with one-carbon less (C n-1 ) as a side reaction in very low yields [24].It is currently unclear how ADO can catalyze one-carbon atom elimination and aldehyde reduction to produce fatty alcohols.
Reaction mechanisms of these enzymes proceed via an oxygen-activation process to generate a reactive radical intermediate.Oxygen binding is indispensable to these enzymes.However, the proposed peroxo intermediates binding mode was predicted based on structural characterization [48] and remains a subject of debate.Isotope labeling studies of the enzyme from Nostoc punctiforme show that one O atom in the formate product is derived from O 2 [34].This result is explained as the attack of the nucleophilic peroxo intermediate on the electrophilic carbon atom of the aldehyde substrate to initially form a peroxy hemiacetal.In support of this mechanism, an intermediate arising from a peroxo band has been trapped [48].Studies of the ADO from Prochlorococcus marinus with C8−10 aldehydes show formation of the expected C7−9 alkane as well as the corresponding primary alcohol and aldehyde in very low yields.Therefore, the role of oxygen in ADO reaction and reaction mechanism of alkane and alcohol products formation are still not clear.
Here, we found a novel activity of PmADO to generate full-length fatty alcohols, not one-carbon less, from aldehydes instead of alkane.The reaction mechanism of this aldehyde reductase activity and the role of oxygen and Fe 2+ cofactor was investigated.We rst explored fatty alcohol production in the metabolic engineered cell carrying the ADO gene.We found that in addition to alkanes, the cell containing ADO generated fatty alcohols in signi cant amount.We investigated in-depth how fatty alcohol can be generated by ADO using the puri ed ADO and the reconstituted system of ADO and redox partners.The results showed that ADO can produce fatty alcohols from fatty aldehydes without requiring O 2 , metals and canonical redox partners such as ferredoxin/ferredoxin reductase (Fd/FNR) or phenazine methosulfate (PMS).Our work is the rst study to report that under O 2 -limited conditions, PmADO exhibits NADPH-dependent reductase activity which mainly catalyzes direct hydride transfer from NADPH to fatty aldehydes to produce fatty alcohols in the full-chain form.It should be noted that this activity is different from the previous results reporting very small amount of one-carbon less fatty alcohol production.The new aldehyde reductase activity of ADO is high and e cient, suggesting it's promising potential in production of fatty alcohols via bio-process in the future.

Production of fatty alcohols by E. coli cells containing ADO
As illustrated in the previous report, alkane production can be produced from the synthetic metabolic pathway in Escherichia coli having aldehyde deformylating oxygenase (ADO) from Prochlorococcus marinus MIT9313, fatty acyl-CoA reductase (ACR1) from Acinetobacter baumannii and associated with redox partners ferredoxin (Fd) from Synechocystis sp.PCC 6803, ferredoxin ( avodoxin): NADP + oxidoreductase (FNR).It was shown that addition of formate dehydrogenase (FDH) from Xanthobacter sp.91 could increase intracellular NADH/NAD + ratio in cells which can increase higher level of reduced Fd and increase yield of alkane production ~50% (23).Here, we further explored why the alkane product was limited at ~50% by further exploring whether this cell can generate other types of by-products.Analysis of metabolite pro les of the engineered cells containing PmADO and various types of auxiliary systems indicated that these cells produced signi cant amount of fatty alcohol, up to 25% fatty alcohols (Figure 1).This result prompted us to explore in-depth reaction mechanisms of fatty alcohol formation by PmADO using the puri ed enzyme and reconstituted systems.

Fatty alcohol production by the puri ed PmADO
Based on the results above showing that the metabolic engineered cell overexpressing PmADO could produce signi cant amount of fatty alcohol, we thus explored aldehyde reductase activity of the puri ed PmADO and also carried out experiments to rule out possible involvement of contaminating aldehyde reductases from the E. coli cell.As shown in Figure 2, the PmADO (with a subunit molecular weight of 24 kDa) obtained from our puri cation process (Experimental Procedures) is highly pure (>98% purity) with no other protein contamination visible by SDS-PAGE analysis.Based on the E. coli genome sequence, we identi ed putative aldehyde reductases produced in cells to monitor whether they could introduce possible false reductase activity in our PmADO assays.The analysis summarized in Additional le 1: Table S1 indicates that other genes identi ed as reductases (yahK, frmA, adhE, adhP, eutG, yqhD, yiaY and yjgB) all have their protein subunit sizes around 35-96 kDa.Results in Figure 2 clearly show that the puri ed PmADO obtained from our preparation has no (not even trace amount) contaminating protein in that region, ruling out a possibility of having other aldehyde reductases present in our enzyme sample.
Using assays described in Experimental Procedures and in Table 1, the results indicated that the puri ed PmADO could catalyze fatty alcohol production from fatty aldehyde using various reducing systems (more results discussed below).

Fatty alcohol production by the unusual reaction of PmADO reductase activity
We rst gured out nature of reductant required for PmADO aldehyde reductase activity and the effect of oxygen on this reaction.Reaction mixtures containing dodecanal and various %O 2 (<0.0004%, 10%, and 15%) in the presence or absence of a reducing system, which are ferredoxin and ferredoxin reductase (Fd/FNR) were analyzed by GC/MS.Amount of fatty acid, alkane and alcohol products produced under various concentrations of oxygen and the reducing systems are shown in Table 1.The alcohol production by PmADO decreased when the O 2 concentration was increased.The highest alcohol production was observed under anaerobic conditions with less than 0.0004% oxygen for both systems of PmADO with NADPH and PmADO with NADPH and Fd/FNR (Table 1).We noted that the alcohol production was not observed in the reactions without NADPH (Additional le 1: Figure S1).
Interestingly, dodecanal could be converted to dodecanol by PmADO in the absence of ferredoxin and ferredoxin reductase.The results showed that alcohol production yield by PmADO with NADPH (69 ± 5 µM) exhibited a similar value of product to the reaction of PmADO with the Fd/FNR reducing system (75 ± 4 µM) at less than 0.0004% O 2 (Table 1).In the presence of the reducing system (Fd/FNR), the PmADO reaction showed an increase in alkane along with acid production when oxygen was increased (Table 1).However, the reaction of PmADO without the ferredoxin and ferredoxin reductase showed only acid in addition to the alcohol product (Table 1).These results indicate that the Fd and FNR are not necessary for transferring electrons from NADPH to produce alcohol by PmADO.Notably, PmADO can convert aldehydes to n-alcohols in the presence of only NADPH.As the highest yield could be obtained under anaerobic conditions, the data suggest that O 2 is not necessary for the production of fatty alcohol by PmADO.Therefore, a minimum system for production of alcohol by PmADO only requires aldehyde and NADPH substrates (Scheme 1).Factors affecting production of fatty alcohol from aldehyde by PmADO including reducing systems and O 2 concentrations are summarized in Table 1 and Figure 3.
To further address the effects of oxygen on production of fatty alcohol and to ne tune conditions for maximum alcohol production, reactions were performed in an anaerobic glove box and pre-incubated with an oxygen scavenging system glucose and glucose oxidase (Glc/GOx) to completely remove oxygen.Under these conditions and using octanal as a substrate, the detected octanol (80% conversion) was 20% greater than that of the reaction without oxygen scavenging system (60% conversion) (Figure 3B).Accordingly, no heptane or octanoic acid could be detected in these reaction mixtures.These data solidly demonstrate the conversion of fatty aldehyde to yield 80% fatty alcohol by aldehyde reductase activity of PmADO using NADPH as an electron donor in the absence of O 2 and other redox partners.
To explore speci city of reducing equivalent for aldehyde reductase activity of PmADO, we investigated whether the enzyme can use NADH in addition to NADPH.The result showed that the PmADO aldehyde reductase activity could only use NADPH not NADH to produce fatty alcohol (Figure 4).Even with increased concentrations of PmADO (20,40,80, and 160 µM) in the presence of NADH, none of the reactions could produce fatty alcohols (Additional le 1: Figure S2).
The above ndings showing that the puri ed PmADO could only use NADPH as a reductant also ruled out a possibility that the aldehyde reductase activity observed in this report comes from contamination of other aldehyde reductases produced in E. coli cells.Most of aldo-keto reductases in E. coli are known to use both NADPH and NADH as reductants to reduce a variety of aldehydes and carbonyl moieties [34,[46][47][48][49] (Additional le 1: Table S1).The only aldehyde reductase in E. coli which can use NADPH as a reductant is yqhD (Additional le 1: Table S1).As a subunit molecular weight of yqhD is 42 kDa, this indicates that the puri ed PmADO with a subunit molecular weight of 24 kDa (Figure 2) has no contamination from yqhD or other aldehyde reductases from E. coli.Altogether, all data suggest that the aldehyde reduction by NADPH was indeed catalyzed by the puri ed PmADO.
A ferrous (Fe 2+ ) ion is not necessary for the aldehyde reductase activity of PmADO The role of metal cofactor in aldehyde reductase activity of PmADO was investigated.We explored the involvement of Fe 2+ -cofactor in this activity by preparing an apoenzyme form of PmADO and measured its activity in comparison with Fe 2+ -bound holoenzyme.Although the native O 2 -dependent ADO reaction is involved with formation of an iron (III)-peroxo intermediate [34,50] ,the aldehyde reductase activity to generate fatty alcohol is not involved with the metal cofactor and may not require the diiron-cofactor.Results from comparison of activities of ferrous supplemented PmADO, holo-PmADO and apo-PmADO (Figure 5) showed that the yield of fatty alcohol production from holo-PmADO (~60%) was similar to that observed in the reaction of apo-PmADO (~55%) and adding extra Fe 2+ to the reaction (~75%).Based on standard variations of measurements, these values are all in the same range.Here, the results indicate that the metal cofactor is not involved in the reductase activity of PmADO.The MD simulations results (discussed later) also con rmed this conclusion.
The reductase activity of PmADO is highly speci c towards medium-to long-chain aldehydes Speci city of aldehydes that can be reduced by PmADO was investigated by carrying out the reactions of PmADO with medium-and long-chain (C 6 , C 8 , C 10 , C 12 , and C 14 ) aldehydes and analyzed for alcohol production (Table 2, Figure 6).Based on kinetic parameters, the results indicate that PmADO does not exhibit strong chain length speci city with aldehyde substrates.This feature is consistent with speci city of aldehyde usage in the native PmADO activity in which both native cyanobacteria and reconstituted PmADO systems in E. coli can generate a wide range of alkane products [25,51].The results show that conversion of decanal to decanol gave the highest yield (98%) among all substrates used.Percentages of other aldehyde conversion to alcohol products were 86%, 79%, 68%, and 70% for hexanal, octanal, dodecanal, and tetradecanal, respectively.k cat and K m values of hexanal, octanal, and decanal are all relatively similar with a moderate trend of decreasing K m with increasing chain length of aldehyde from C 6 to C 10 (10.8 µM for C 6 , 10.3 µM for C 8 , and 6.5 µM for C 10 (Table 2)).On the contrary, for the case of C 12 and C 14 , the K m value increases with increasing chain length (Table 2); k cat of C 12 is higher than that of C 14. Altogether, these trends contribute to an approximately 3-to 5-fold changes in reaction e ciency (k cat /K m ) from 15 × 10 -3 μM −1 min −1 for C 10 to 3 × 10 -3 μM −1 min −1 for C 12 and 5 × 10 -3 μM −1 min −1 for C 14 (Table 2).It is important to note that no alcohol production was observed when 2-octenal was used as a substrate (Additional le 1: Figure S3).

Molecular docking and molecular dynamics (MD) simulations to identify a putative NADPH binding site
In order to identify a putative NADPH binding site in PmADO, MD simulations was used to dock NADPH and aldehyde into the PmADO active site.As decanal gave the highest catalytic e ciency in alcohol production by PmADO (Table 2), decanal was chosen to use in molecular docking and MD simulations studies.A structure of decanal was generated based on the structure of 11-[2-(2-ethoxyethoxy) ethoxy undecanal, which could be co-crystallized as a ligand in the PmADO crystal structure (PDB code 4PGK).NADPH was placed into the system containing PmADO and decanal.MD simulations were used to release stain of the protein and explore NADPH binding at the active site with the lowest energy point (-8.4 kcal/mol).The results showed that NADPH could bind to helices 1, 2, 4, and 5, which are close to the aldehyde binding site of PmADO.Snapshots taken around 0.54 ns of MD simulations are shown in Figure 7A.Moreover, the MD simulations also indicated that distances between C α of Glu73 and C α of Glu157 changed over the course of MD simulations, representing open and close forms of the PmADO enzyme.The open form could be observed before 0.54 ns (C α of Glu73 and C α of Glu157 bond distance increased from 12 to 20 Å) while the semi-closed form occurred after 0.54 ns (C α of Glu73 and C α of Glu157 bond distance decreased from 20 to 14 Å) (Figure 7B).These dynamics may represent the PmADO:NADPH:aldehyde complex conformational change prior to the next reduction step.
Additionally, the MD simulations of the PmADO:NADPH: decanal complex identi ed the distance between the C4-position of hydrogen atom of NADPH and the carbonyl carbon of fatty aldehyde (Figure 7A) as 3.8 Å.With this binding mode, the enzyme-bound NADPH can make stable contacts with decanal in the binding site of the PmADO.It should be mentioned that the PmADO structure used in our MD simulations (PDB code: 4PGK) was solved under aerobic conditions which may not represent the structure relevant to aldehyde reductase activity reported here.A hydride transfer distance in this structure may not be close enough for aldehyde reduction to occur.Up to now, no anaerobic crystal structure of an ADO enzyme is available.In the presence of molecular oxygen, the active site might be blocked, and consequently prohibiting the reaction between NADPH and aldehyde.In the case of aldehyde reductase, it is known that the hydride transfer from a nicotinamide co-substrate occurs only with a dehydrated aldehyde [52].As aldehyde in solution likely exists in the hydrated form, thus during binding of a substrate, water must be stripped away for the substrate to prompt the compound for the catalysis [53].This phenomenon explains the superiority of anaerobic alcohol production by ADO enzyme because molecular oxygen was removed for a proper binding of the enzyme substrate complex.The unique activity of PmADO that directly reduces fatty aldehyde by NADPH under anaerobic conditions provides structural and functional insights of PmADO for future biotechnology applications.

The reductase activity of recombinant PmADO enhances alcohol production in the metabolically engineered cell
To demonstrate the fatty alcohol production from reconstituted PmADO in E. coli under limited oxygen concentration, the PmADO and carboxylic acid reductase from Mycobacterium marinum (MmCAR) were constructed and measured fatty alcohol production in the metabolic engineered cell.MmCAR can catalyze fatty acid to aldehyde.Then, PmADO can use fatty aldehyde as a substrate to produce fatty alcohol.In order to avoid complications, decanoic acid was used as a supplement.The plasmids consisting of only MmCAR were also constructed to investigate the native aldehyde reductase activity from endogenous aldehyde reductase (AHR) in E. coli.Both plasmid systems were expressed in E. coli and the production of alcohol was quanti ed by GC/MS after 6 hours of a bioconversion process under various oxygen concentrations (<0.0004, 5, 10, 15, 20%).The results showed that fatty alcohol production could be observed in both MmCAR and MmCAR+PmADO under all oxygen concentrations (Figure 8).However, fatty alcohol production in the cell harbouring MmCAR+PmADO showed much greater amount of product than that of MmCAR cell factory, approximately 2-fold under almost all oxygen concentrations.The exception was found for the condition with <0.0004% O 2 because both cell types showed low alcohol production (22%).This might be due to the disruption of energy metabolism in E. coli cells under strict anaerobic conditions [54].Under 5% oxygen, MmCAR+PmADO gave the highest alcohol yield of dodecanol which is about 75%.This nding demonstrates that PmADO has the ability to enhance alcohol production in a cell factory as well as in vitro.

Discussion
Our work here found a new activity of PmADO which has not been documented.PmADO was reported to convert aldehyde to alkane or alkene under aerobic conditions [24], and alcohol products with one-carbon less could also be generated.ADO typically requires a reducing system to produce alkane or alkene [50].
However, when the reaction contains extra O 2 , the reaction sometimes proceeds through the formation of acid in addition to alkane [50].The previous study has shown that ADO under fully aerobic conditions showed comparatively low activity.However, under micro-aerobic (3-10% O 2 ) conditions, ADO could carry out the reaction for maximum of 3 turnovers with dodecanal as a substrate [55].Moreover, O 2 is strictly required for bioconversion to produce the n-1 carbon alkane products along with n-1 aldehyde and n-1 alcohol.Previously, it was shown that only 2% of heptanol could be produced from octanal [24].The system strongly requires O 2 to produce n-1 aldehyde and n-1 alcohol because of the reaction mechanism involved [24].Under our investigation, the reactions which were run in an anaerobic chamber following preincubation with oxygen scavenging system also did not detect any n-1 alcohol products, consistent with the previous nding.However, we detected full-length alcohol products which were not previously reported.
In this context, our ndings here of full-length fatty alcohol products and that PmADO does not require dioxygen or canonical reductant (Fd/FNR) nor di-iron cofactor for alcohol production but only requires a hydride transfer from NADPH were unexpected.The results obtained prompted us to explore the peculiar activity of PmADO in fatty aldehyde reduction by a direct hydride transfer process under anaerobic conditions.We found that PmADO could convert aldehydes into full-length alcohols up to 80% yield under a condition containing O 2 -scavenging system.Analysis by MD simulations of NADPH binding with PmADO also support the binding of this ligand (Figure 7).The substrate-binding site of ADO comprises a long hydrophobic channel that terminates at the di-iron centre [55].Similar to other aldehyde reductase (AHR) crystal structues, the binding cavity comprises hydrophobic pockets which can accommodate a nicotinamide ring and aldehyde substrate [56].Recently, the active-site residues have been identi ed and showed that the residues close to the di-iron centre exerted in uence on ADO activity [57].
This novel reduction reaction is also different from the native deformylation reaction of PmADO on the substrate speci city.The reduction reaction shows almost full conversion (98%) only with medium chain aldehydes.In the case of deformylation reaction, it shows broad substrate speci city in all long-and short-chain aldehydes; octadecanal and heptanal, were used as substrates for ADO almost equally well [25, 28, 29, 33-36, 58, 59].Based on the co-crystal structures of cyanobacterial ADO with substrate analogues [22,30], changes of the amino acids close to the aldehyde binding site and the hydrophobic tail of the substrate and those along the substrate channel may affect the substrate speci city [60].Moreover, the structures of ADO demonstrated that several modes of binding are possible for aldehydes.In the case of short-chain aldehydes, more than one molecule has been proposed to bind in the ADO substrate cavity [24].These results suggest that non-productive substrate binding modes might be present.It was, then, hypothesized that the location of NADPH might be a key factor to increase mediumchain aldehyde interaction in PmADO active site and promote the hydride transfer, which is supported by the highest k cat /K m of 250 M −1 s −1 with 98% conversion of decanol.The biosynthesis of fatty alcohol by thioesterases has been investigated in the production of various chain length alcohols [1,[17][18][19]32].However, the aldehyde reductase, which is responsible for reduction of speci c chain length aldehydes to alcohol, remains unclear.In the case of yjgB [32] and yqhD [1], these enzymes demonstrated improvement in long-chain alcohol production.Nevertheless, the enzymes that play major roles for conversion of long-chain aldehyde into alcohol is yet to be clari ed.Therefore, the knowledge obtained from this study will be useful in designing new e cient biocatalysts for the production of fatty alcohols.
Previous studies of the metabolic engineering for alkane biosynthesis using ADO all showed that the alkane production yield was less than 50% [12,16,23,32,[61][62][63][64].This might be due to the fact that ADO could produce both alkane and fatty alcohol, diverting the yield of alkane biosynthesis.Apart from production by ADO, the metabolic engineered cells containing aldehyde producing enzymes can also generate fatty alcohols from endogenous aldehyde reductases (Figure 1) [8,16,62].Our studies demonstrated that yield of fatty alcohol products in whole cell biocatalysis can be affected by oxygen concentration.Under 10-20% O 2 , yields of decanol was around 60-70% and could be increased up to 75% under 5% O 2 which is 58% higher than that of endogenous aldehyde reductase strain.Therefore, the metabolically engineered cells such as those demonstrated in Figure 8 can be employed as biocatalysts with dual and tunable activities.Our knowledge here suggests that in the future, the same cells can be tuned for production of alkane or fatty alcohol depending on bioconversion conditions.

Conclusion
In this work, we have shown for the rst time that PmADO exhibits unusual activity to reduce fatty aldehydes to fatty alcohols by a reduction process using only NADPH as an electron donor under O 2limitted conditions.This feature makes PmADO a more versatile catalyst than previously recognized.It is possible to use the enzyme for alcohol production by providing the enzyme with aldehyde and NADPH under anaerobic conditions.This phenomenon requires NADPH to donate a hydride to aldehyde to generate alcohol, in which up to 80% fatty alcohol could be produced in the reactions containing O 2scavenger system while supplementing Fe 2+ to the PmADO reaction showed no signi cant impact on alcohol production.PmADO revealed a wide range of aldehydes that can be used as substrates.The activity suitable for the medium-chain aldehyde usage could yield up to 98% conversion and showed very high k cat /K m (250 M −1 s −1 ).Moreover, MD simulations con rmed that NADPH can bind to the active site of the PmADO, which is close to the aldehyde binding site with binding energy of -8.4 kcal/mol.The MD simulations results also showed that NADPH has stable contacts with aldehyde in the binding site of the PmADO.Moreover, PmADO can enhance the alcohol production in a cell factory, making the cell having dual activities for both alkane and alcohol production.These ndings open a new biocatalytic application of PmADO in terms of fatty alcohols production.

Chemicals and reagents
All chemicals were commercially available and of high quality and analytical grade.Oligonucleotides and the gene encoding ADO from Prochlorococcus marinus (strain MIT 9313) (pmado) were synthesized and codon optimized by the GenScript Biotech Corp. (China).Restriction endonucleases NdeI and BamHI were from New England BioLabs.
In vivo fatty alcohol production by whole-cell biocatalysis E. coli BL21 (DE3) cells harboring each set of gene constructs were used for alkane and fatty alcohol production (pET17b-acr1, pCDFDuet-1-fd-fnr-pmado, pRSFDuet-1-trxA-trxB and pRSFDuet-1-fdh).To determine the ability of PmADO to produce alcohol, cells containing pET17b-mmcar/pCDFDuet-1-pmado and pET17b-mmcar were cultured in 5 mL of LB medium contain 100 µg/ml ampicillin or 25 µg/ml streptomycin at 37 °C for 16 hours.A 1% (v/v) inoculum of bacterial culture was further cultured in 150 mL of terri c broth at 37 °C.Until OD 600 of the culture reached about 0.5, the temperature of the culture medium was lowered to 25 °C.Then, 1 mM isopropyl-b-D-glucopyranoside (IPTG) was added and the culture was maintained at this temperature for 8 hours.The cell paste was resuspended and adjusted cell density to OD 600 of 20 in 100 mM potassium phosphate buffer pH 7.5 supplemented with 2.5% glucose, and 0.85 mM tetradecanoic acid.
In experiments with various oxygen concentrations, the harvested cell paste was resuspended inside an anaerobic glover box and adjusted cell density to OD 600 of 20 in 100 mM potassium phosphate buffer, pH 7.5 containing 0.8 mM decanoic acid.The resuspension was adjusted with various O 2 concentration (<0.0004, 5, 10, 15 and 20%).Then, the suspension was incubated at 25 °C for 6 hours.Samples were collected, extracted by ethyl acetate separation, and analyzed by GC/MS for quantitation of decanol product.

Enzyme expression and puri cation
Escherichia coli BL21(DE3) cells harbouring pET17b-pmado were cultured in a ZYM-5052 auto-induction rich medium containing 50 μg/mL of ampicillin, and 1X 5052 medium at 37 °C until OD 600 of the culture reached approximately 1.Then, the temperature of the culture medium was adjusted to 25 °C and maintained at this temperature for 6-8 hours to overexpress PmADO.PmADO was puri ed to homogeneity using precipitation methods (0.5%(w/v) PEI and 40-60%(w/v) (NH 4 ) 2 SO 4 precipitation) and anion-exchange column chromatography (DEAE-Sepharose).The purity of PmADO after puri cation was analyzed by 12%(w/v) SDS-PAGE, which identi ed its subunit molecular weight as 24 kDa (Figure 2).Most of molecular weights of endogenous aldehyde reductases commonly found in E. coli BL21(DE3) are larger (35-96 kDa) than 24 kDa.
For overexpression of the reducing systems (Fd and FNR), the expression constructs of pCDFDuet-1 plasmid conjugated with the fd gene from Synechocystis sp.PCC 6803 and modi ed with 6xHis on Nterminus (pCDFDuet-1-fd) and the fnr gene from E. coli K-12 (pCDFDuet-1-fnr) were overexpressed in E. coli BL21(DE3) cells cultured in LB medium containing 25 μg/mL streptomycin at 25 °C for 6-8 hours with the addition of 1 mM IPTG to induce protein production.Fd was puri ed to homogeneity using precipitation methods (0.1%(w/v) PEI and 0-10%(w/v) (NH 4 ) 2 SO 4 precipitation) and a nity column chromatography (Nickel-chelating Sepharose).The purity of Fd after puri cation was analyzed by 14% (w/v) Tricine SDS-PAGE which determine its subunit molecular weight as 9.4 kDa.FNR was puri ed to homogeneity using precipitation methods (1%(w/v) PEI and 40-80%(w/v) (NH 4 ) 2 SO 4 precipitation) and anion-exchange column chromatography (DEAE-Sepharose).The purity of FNR after puri cation was analysed by 12%(w/v) SDS-PAGE, which determine its subunit molecular weight as 27 kDa.

Activity assay and product analysis
Activity assays were performed in 100 mM HEPES buffer, pH 6.8, containing 100 mM KCl. Aldehyde substrates (C 6-14 ) were prepared as a 10 mM stock solution in methanol.A typical assay contained 10 µM PmADO, 250 µM aldehyde substrate and 1 mM NADPH.To study the effect of the reducing system on alcohol production by PmADO, 10 µM Fd, and 10 µM FNR were added into the reaction.The assay reactions were placed at 37 °C, quenched by addition ethyl acetate in a 1:1 ratio, and vigorously vortexed before centrifugation at 13,800 ´g and 4 °C for 10 min.The clear organic phase was analysed by gas chromatography/mass spectrometry (GC/MS) using a HP-5 column, and the column was continuously own with a He gas at a constant ow rate of 7 ml/min and 250 °C injection port.The sample was split at the mass spectrometer (GC/MS) using the following oven temperature programme: 60 °C held for 3 minutes, 200 °C held for 2 min, and 260 °C held for 3 min at 10 °C min −1 and 20 °C min −1 .Concentrations of products were determined by a standard curve.

Effects of oxygen and the reducing system on alcohol production by PmADO
The effects of oxygen on the PmADO activity were assessed inside an anaerobic glove box (<0.0004% O 2 , Belle Technology, UK) to avoid the disturbance of oxygen in air.The alcohol product yielded by PmADO reaction containing various concentrations of oxygen, <0.0004 (mostly anaerobic), 10, and 15%, and in the absence and presence of the reducing systems (Fd/FNR) was analysed by GC/MS as described above.To deplete oxygen completely, the reaction buffer was pre-incubated with an oxygen-scavenging system, 200 µM glucose and 10 µg/ml glucose oxidase (Glc/GOx) before the reaction started.

Effect of metal cofactor on alcohol of PmADO
The metal contents in the puri ed PmADO were determined using an inductively coupled plasma optical emission spectrometer (ICP-OES).Apoenzyme of PmADO (apo-PmADO) was prepared by either incubating PmADO with a 5-fold excess concentration of EDTA or treating the enzyme solution with Chelex 100 chelating gel (Bio-Rad Laboratories, Inc., USA) overnight, and the EDTA-metal complexes were removed by a PD-10 (Sepahdex G-25) desalting column pre-equilibrated with 100 mM HEPES buffer, pH 6.8, containing 100 mM KCl that was prepared from a Milli-Q ® Type 1 ultrapure water.The eluted apoenzyme was collected, and the protein concentration of apo-enzyme was determined at 280 nm (ε= 17,545 M -1 cm -1 ).

Table 2 .
Substrate speci city 70.PhillipsJ, Isgro T, Sotomayor M, Villa E, Yu H, Tanner D, et al.NAMD tutorial.2012.71.Isgro T, Phillips J, Sotomayor M, Villa E, Yu H, Tanner D, et al.NAMD tutorial.2017.Tables Table of the corresponding alkane, acid, and alcohol products obtained from dodecanal conversion by PmADO catalysis under various %O 2 in the presence and absence of reducing partner.Kinetic parameters of PmADO activity towards C 6 , C 8 , C 10 , C 12 , and C 14 Aldehydes k cat (min -1 ) K m (µM) k cat /K m (µM -1 min -1 ) aThe product formed with one carbon less (C n-1 ).bThe product formed with a full-chain length.N.D., not detectable