Dehydrogenation Mechanism of Three Stereoisomers of Butane-2,3-Diol in Pseudomonas putida KT2440

Pseudomonas putida KT2440 is a promising chassis of industrial biotechnology due to its metabolic versatility. Butane-2,3-diol (2,3-BDO) is a precursor of numerous value-added chemicals. It is also a microbial metabolite which widely exists in various habiting environments of P. putida KT2440. It was reported that P. putida KT2440 is able to use 2,3-BDO as a sole carbon source for growth. There are three stereoisomeric forms of 2,3-BDO: (2R,3R)-2,3-BDO, meso-2,3-BDO and (2S,3S)-2,3-BDO. However, whether P. putida KT2440 can utilize three stereoisomeric forms of 2,3-BDO has not been elucidated. Here, we revealed the genomic and enzymic basis of P. putida KT2440 for dehydrogenation of different stereoisomers of 2,3-BDO into acetoin, which will be channeled to central mechanism via acetoin dehydrogenase enzyme system. (2R,3R)-2,3-BDO dehydrogenase (PP0552) was detailedly characterized and identified to participate in (2R,3R)-2,3-BDO and meso-2,3-BDO dehydrogenation. Two quinoprotein alcohol dehydrogenases, PedE (PP2674) and PedH (PP2679), were confirmed to be responsible for (2S,3S)-2,3-BDO dehydrogenation. The function redundancy and inverse regulation of PedH and PedE by lanthanide availability provides a mechanism for the adaption of P. putida KT2440 to variable environmental conditions. Elucidation of the mechanism of 2,3-BDO catabolism in P. putida KT2440 would provide new insights for bioproduction of 2,3-BDO-derived chemicals based on this robust chassis.


Bacteria and Culture Conditions
Bacterial strains and plasmids used in this work are listed in Supplementary Table S1. P. putida KT2440 and its derivatives were cultured in MSM (Ma et al., 2007) with 2 g L −1 different substances at 200 rpm and 30°C. Lanthanide concentrationdependent growth of P. putida KT2440 was monitored using a Bioscreen microbiology reader (Bioscreen C Labsystems, Helsinki, Finland). E. coli DH5α and BL21 (DE3) were cultured in Luria-Bertani (LB) medium at 180 rpm and 37°C. Antibiotics were added to the medium when necessary, at the following concentrations: kanamycin at 50 μg mL −1 and ampicillin at 100 μg mL −1 .

Reverse Transcription-Polymerase Chain Reaction
RT-PCR experiments were conducted as described previously (Zhang et al., 2018). Total RNA was isolated from P. putida KT2440 cells grown to mid-log phase in MSM supplemented with different carbon sources using Qiagen RNeasy total RNA Kit. DNA contamination was eliminated by RNase-free DNase I treatment. cDNA was generated using Superscript II RT Kit. Samples were incubated at 25°C for 10 min and 42°C for 30 min, then heated at 85°C for 5 min. RT-PCR was performed with Taq DNA Polymerase (Transgen, China) using corresponding oligonucleotides (Supplementary Table S2). The genomic DNA and total RNA of P. putida KT2440 were used as positive and negative controls, respectively.

Expression and Purification of R,R-BDH
The gene encoding R,R-BDH was amplified from genomic DNA of P. putida KT2440 using primers pp0552-F/pp0552-R (Supplementary Table S2). The PCR product was cloned into the pETDuet-1 plasmid to obtain plasmid pETDuet-pp0552. E. coli BL21(DE3) containing pETDuet-pp0552 was grown to an optical density at 600 nm (OD 600nm ) of 0.6-0.8 in LB medium with 100 μg mL −1 ampicillin at 37°C, 180 rpm and induced at 16°C with 1 mM isopropyl-D-1-thiogalactopyranoside (IPTG). The cells were harvested, washed twice, and resuspended in buffer A (pH 7.4, 20 mM sodium phosphate and 500 mM sodium chloride), then lysed by sonication in an ice bath after the addition of 1 mM phenylmethanesulphonyl fluoride (PMSF) and 10% glycerol (vol/vol). The cell lysate was centrifuged at 12,000 × g for 40 min at 4°C to remove cell debris. The supernatant was loaded onto a HisTrap HP column (5 mL) and eluted with buffer B (pH 7.4, 20 mM sodium phosphate, 500 mM sodium chloride, and 500 mM imidazole). The quality of purified protein was analyzed by sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE) with 12.5% polyacrylamide gels and the concentration was determined by the Bradford protein assay Kit.

Enzymatic Assays of R,R-BDH
The activity of R,R-BDH was assayed by measuring the change in absorbance at 340 nm at 30°C. For assay of the oxidative activity, the reaction solution contained 10 mM substrates and 1 mM NAD(P) + in Tris-HCl buffer (pH 7.4, 50 mM). For assay of the reductive activity, the reaction solution contained 5 mM substrates and 0.2 mM NAD(P)H. One unit of enzyme activity was defined as the amount of enzyme that consumed or produced 1 μmol of NAD(P)H per min.
To determine the reductive product of the R,R-BDH, the 1 ml reaction solution containing 1 mM AC or diacetyl, 1 mM NADH, 10 U purified R,R-BDH in Tris-HCl (pH 7.4, 50 mM) was incubated at 30°C and 180 rpm for 1 h. The solutions were centrifuged at 13,000 × g for 10 min and the supernatant was used for detection. The concentrations of 2,3-BDO and AC were analyzed by a gas chromatography system (Varian 3800, Varian) equipped with a flame ionization detector and a 30-m SPB-5 capillary column (0.32-mm inner diameter, 0.25-μm film thickness; Supelco, Bellefonte, PA) using nitrogen as the carrier gas. The injector and detector temperatures were 280°C while the column oven temperature was maintained at 40°C for 3 min and then raised to 240°C at a rate of 20°C min −1 (Ma et al., 2009;Xu et al., 2014). The stereo configuration of 2,3-BDO produced from AC or diacetyl was detected by a gas chromatography system (Agilent GC6820) equipped with a flame ionization detector and a fused silica capillary column (Supelco Beta DEX TM 120, inside diameter, 0.25 mm; length, 30 m) using nitrogen as the carrier gas. The injector and detector temperatures were 280°C while the column oven temperature was maintained at 40°C for 3 min, raised to 80°C at a rate of 1.5°C min −1 , then increased to 86°C at a rate of 0.5°C min −1 and finally raised to 200°C at a rate of 30°C min −1 (Xiao et al., 2010).

Construction of P. putida KT2440 Mutants
To construct the Δpp0552 mutant strain of P. putida KT2440, the upstream and downstream homologous arms of the pp0552 gene were amplified using the primers pp0552-up-F/R and pp0552-down-F/R, respectively (Supplementary Table S2). Homologous arms were fused together via recombinant PCR and amplified with the primers pp0552-up-F and pp0552-down-R. The product was inserted into a mobilizable plasmid pK18mobsacB (Schäfer et al., 1994), which does not replicate in P. putida, to construct pK18mobsacB-Δpp0552. Then the plasmid was transformed into P. putida KT2440 by electroporation and integrated into the chromosome. The correct single-crossover mutants were selected on LB plate supplemented with 50 μg mL −1 kanamycin. The double-crossover allelic exchange mutant strains were screened on LB plates containing 10% (wt/vol) sucrose. The pedH and pedE mutants of P. putida KT2440 were constructed by using the same procedure.

Utilization of Three Stereoisomers of 2,3-BDO by P. putida KT2440
Rhizosphere is a natural habitat of different microorganism. Some rhizobacteria such as Aerobacter spp., Bacillus spp., and Serratia spp., can produce three stereoisomers of 2,3-BDO as the volatile compounds to trigger induced systemic resistance and promote plant growth (Ryu et al., 2003;Han et al., 2006;Taghavi et al., 2010). P. putida KT2440 is a widely studied rhizosphere-dwelling microorganism. Growth of P. putida KT2440 in minimal salt medium (MSM) supplemented with different stereoisomers of 2,3-BDO was firstly studied. As shown in Figure 1, P. putida KT2440 grew robustly in all three 2,3-BDO stereoisomers with the consumption of each substrate.
Since pp0555, pp0554, pp0553 and pp0552 were located adjacent to acoX (pp0556) and acoR (pp0557) gene homologues of AC utilization operons in other bacteria, we hypothesized that these genes may also comprise a 2,3-BDO utilization operon in P. putida KT2440 (Figure 2A). The mRNA from cells of P. putida KT2440 grown in 2,3-BDO was extracted and reverse-transcribed into cDNA as template for reverse transcription-polymerase chain reaction (RT-PCR). As shown in Figure 2B, the five genes including pp0556, pp0555, pp0554, pp0553 and pp0552 were co-transcribed and might be regulated by AcoR.
The growth of P. putida KT2440 (ΔpedE) in (2S,3S)-2,3-BDO was exclusively stimulated by increased concentrations of lanthanum. In contrast, the growth of P. putida KT2440 (ΔpedH) in (2S,3S)-2,3-BDO was inhibited by lanthanum in a concentration-dependent manner. These results were due to the inverse regulation of PedE and PedH by lanthanide availability, an interesting phenomenon called as lanthanide-mediated switch in several methylotrophic organisms and P. putida KT2440 (Farhan Ul Haque et al., 2015;Vu et al., 2016;Wehrmann et al., 2017;Wehrmann et al., 2018). Since low concentrations of lanthanum could still inhibit the growth of P. putida KT2440 (ΔpedH) in (2S,3S)-2,3-BDO at the presence of Ca 2+ , the lanthanide-dependent PedH seems to be the preferred PQQ-EDH when both metals are simultaneously available Vu et al., 2016;Wehrmann et al., 2017). In addition, the functional redundancy and inverse regulation of PQQ-EDHs provides a mechanism through which P. putida KT2440 can optimize its growth according to the availability of lanthanide under variable environmental conditions (Markert, 1987;Aubert et al., 2002).
P. putida KT2440 can colonize rhizospheres of different plants and induce changes in root exudation of plants like Arabidopsis (Matilla et al., 2010). Interestingly, plant root exudates also regulate expression of genes for 2,3-BDO catabolism and synthesis of some antifungal compounds in rhizosphere Pseudomonas (Mavrodi et al., 2021). As an extracellular metabolite, 2,3-BDO can act as a signaling molecule influencing the action of various organisms in symbiotic habitat of 2,3-BDO producing strains. For example, 2,3-BDO can be a kairomone or pheromone attracting insects and influencing insect propagation (Rochat et al., 2000;Moore et al., 2002). P. putida KT2440 can use endogenous chemical signals to coordinate the expression of various genes (Espinosa-Urgel and Ramos, 2004). Whether 2,3-BDO acts as a signaling molecule of P. putida KT2440 like the situation in insects needs further research. In addition, diverse mechanisms for dehydrogenation of three 2,3-BDO stereoisomers in P. putida KT2440 were ascertained in this work, whether and how the three isomers exhibit different signaling functions also deserve intensive investigation.
In summary, we proved here that P. putida KT2440 is able to utilize all three stereoisomers of 2,3-BDO for growth and revealed the dehydrogenation mechanisms of these stereoisomers. Catabolism of 2,3-BDO generates two molecules of acetyl-CoA without carbon loss  and thus 2,3-BDO is an ideal substrate for the production of chemicals using acetyl-CoA as the precursor. Chemical production of 2,3-BDO based on nonrenewable resources often results in a mixture of three stereoisomeric forms. Many microorganisms have been used to efficiently produce 2,3-BDO but most of them also produce a mixture of two or three stereoisomers of 2,3-BDO. The results reported in this work thus also provide a molecular basis for efficient utilization of chemical or biological produced 2,3-BDO by P. putida KT2440.

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

AUTHOR CONTRIBUTIONS
YL, XW, ML, and LM performed experiments. YL, CL, and CM wrote the manuscript and conceived the study. CL, YL, XW, ML, LM, and WZ were involved in analysis and interpretation of experimental data. CG, CM, and PX coordinated the project.

FUNDING
This work was supported by the grants of National Key R&D Program of China (2019YFA0904900 and 2019YFA0904803), Natural Science Foundation of Shandong Provincial (ZR2018PC008 and ZR2020MC005), Tianjin Synthetic Biotechnology Innovation Capacity Improvement Project (TSBICIP-KJGG-005). The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.