Engineering xylose metabolism for production of polyhydroxybutyrate in the non-model bacterium Burkholderia sacchari
- Linda P. Guamán†1, 2, 3,
- Carlos Barba-Ostria†2, 4, 5,
- Fuzhong Zhang5,
- Edmar R. Oliveira-Filho2View ORCID ID profile,
- José Gregório C. Gomez2 and
- Luiziana F. Silva2Email author
© The Author(s) 2018
Received: 21 March 2018
Accepted: 5 May 2018
Published: 15 May 2018
Despite its ability to grow and produce high-value molecules using renewable carbon sources, two main factors must be improved to use Burkholderia sacchari as a chassis for bioproduction at an industrial scale: first, the lack of molecular tools to engineer this organism and second, the inherently slow growth rate and poly-3-hydroxybutyrate [P(3HB)] production using xylose. In this work, we have addressed both factors.
First, we adapted a set of BglBrick plasmids and showed tunable expression in B. sacchari. Finally, we assessed growth rate and P(3HB) production through overexpression of xylose transporters, catabolic or regulatory genes. Overexpression of xylR significantly improved growth rate (55.5% improvement), polymer yield (77.27% improvement), and resulted in 71% of cell dry weight as P(3HB).
Lignocellulose consisting of cellulose, hemicellulose, and lignin, is the most abundant renewable resource worldwide . Utilization of xylose, the second most abundant sugar in hemicellulosic materials, is essential for the efficient conversion of biomass to high-value chemicals. Some bacteria are known to efficiently transform xylose to polyhydroxyalkanoates (PHA), however, the rates and yields need to be improved to make this biopolymer production economically viable. One potential option to accomplish this is to integrate PHA production to a sugarcane mill [2, 3]. PHAs are accumulated as intracellular granules by several bacterial strains when a carbon source is provided in excess with respect to other essential nutrients (e.g., N, P, K, Mg, Fe, among others) . These renewable biopolymers display properties (biodegradability, biocompatibility, low cytotoxicity) that make them ideal substitutes for oil derived plastics [5–11]. Strategies to improve microbial production using xylose often include bioprocess optimization , overexpression of xylose transporters [13, 14] or catabolic proteins [15, 16]. In recent years, the application of synthetic biology approaches in microbial platforms has opened new possibilities for engineering, optimization [17, 18] or even the re-design of metabolic pathways in Escherichia coli, Saccharomyces cerevisiae, and other model organisms. BglBrick vectors, one of the most widely used expression platforms in synthetic biology, have been used successfully to engineer native and synthetic pathways allowing the production of biofuels, bioplastics precursors, pharmaceuticals and other high-value chemicals [19, 20]. Despite these significant advances, the potential for production at industrial scale is still limited by the use of expensive raw materials, precursors, or the conversion of non-renewable feedstocks, among other factors.
Burkholderia sacchari LMG 19450 LFM101 is a Gram-negative bacterium, isolated from sugarcane crops in Brazil [21, 22], with potential for industrial-scale production of high-value molecules (e.g., xylonic acid, xylitol, and poly-3-hydroxybutyrate [P(3HB)]) from glucose, sucrose, xylose, arabinose, and other renewable carbon sources [12, 23]. Additionally, this bacterium can accumulate up to 80% cell dry weight as P(3HB) from sucrose  and produce hybrid PHA copolymers (incorporating hydroxyalkanoate monomers other than 3-hydroxybutyrate) [24–26]. Despite this great potential, the lack of molecular tools available for this organism and the inherently slow growth rate on xylose (0.15 h−1) must be improved to allow its use for industrial-scale production . In the present study, two BglBrick plasmids were successfully adapted to control protein expression in the non-model bacterium B. sacchari. The constructed plasmids were used to individually overexpress all transporters (xylE and xylFGH), catabolic (xylA and xylB) and regulatory (xylR) genes, involved in xylose utilization through the xylose isomerase pathway, and to evaluate its individual effects on growth rate and production of P(3HB). Expression of appropriate levels of the xylose transcriptional regulator (xylR) enhanced both growth rate and P(3HB) production using xylose as the only carbon source. To our knowledge, this paper represents the first successful attempt to improve growth rate and P(3HB) through overexpression of catabolic and regulator genes in B. sacchari. This work emphasizes the value of developing genetic tools which allow precise and tunable control of expression in non-model organisms.
Materials and methods
Chemicals and media
Unless otherwise specified, all chemicals were obtained from Sigma-Aldrich® (Sigma-Aldrich, Saint Louis, Missouri, USA). Luria–Bertani medium (10 g/L tryptone, 10 g/L NaCl, and 5 g/L yeast extract, pH 7.4) was used for cloning purposes. Minimal medium (MM) used for growth and P(3HB) accumulation assays was modified from  and contains in g/L: KH2PO4 (0.39); (NH4)2SO4 (2.91); MgSO4·7H2O (0.31); CaCl2·2H2O (0.010); (NH4)5Fe(C6H4O7)2 (0.06); NaCl (1); trace elements solution (2 mL/L), which was prepared with H3BO3 (0.30 g/L); CoCl2·6H2O (0.20 g/L); ZnSO4·7H2O (0.10 g/L); MnCl2·4H2O (0.03 g/L); NaMoO4·2H2O (0.03 g/L); NiCl2·6H2O (0.02 g/L); CuSO4·5H2O (0.01 g/L). Use of xylose or glucose as carbon sources for MM is indicated as MMX and MMG, respectively. Sterile filtered solutions of arabinose or isopropyl β-d-1-thiogalactopyranoside (IPTG) were used as inducers at the indicated concentrations.
Bacterial strains and growth conditions
Escherichia coli DH10B (F− endA1 deoR+ recA1 galE15 galK16 nupG rpsL Δ(lac)X74 φ80lacZΔM15 araD139 Δ(ara,leu)7697 mcrA Δ(mrr-hsdRMS-mcrBC) StrR λ−) was used as a host for plasmid construction and propagation . Escherichia coli cultures were grown in Luria–Bertani broth (200 rpm) or agar at 37 °C. When needed, the medium was supplemented with kanamycin (kan, 50 μg/mL). Burkholderia sacchari LMG 19450 LFM101 was used as the final host for pF8k-RFP and pF5k-RFP plasmids. Cells were grown at 30 °C, 150 rpm in MMG for pre inoculum to achieve higher biomass to begin the assay and MMX (5 g/L) for 24 h for growth assays and 15 g/L for 48 h for P(3HB) accumulation.
Bioinformatic analysis and primer design
Primers designed in the present study to amplify the genes of interest for cloning in the compatible BglBrick plasmid
Genes were amplified from the B. sacchari genome, using Q5® High-Fidelity DNA Polymerase (New England Biolabs, Inc, Ipswich, Massachusetts, USA) in a Mastercycler Nexus Gradient model thermal cycler (Eppendorf AG, Hamburg, Germany) following the protocol recommended by the manufacturer. Cycling conditions were as follow: 98 °C 2′, 35X [98 °C 20″, 60 °C 20″, 72 °C (25″ per Kb)], 72 °C 5′.
Plasmids used in the present study
Burkholderia sacchari transformation
Electrocompetent cells of B. sacchari were prepared according to previously established protocols. Briefly, 1 mL of culture in mid-exponential phase was inoculated into 50 mL of Luria–Bertani (250 mL flask) and incubated at 30 °C at 170 rpm until cells reached OD600 0.6. The flask was incubated 15 min on ice, collected by centrifugation (15 min, 4500 rpm), washed twice with 50 mL of sterile water. The cell pellet was finally resuspended and aliquoted (50 µL each) in sterile 20% glycerol. Plasmids were transformed by electroporation using Gene Pulser Xcell™ electroporation system (Bio-Rad Laboratories, Inc. Hercules, California, USA). 100 ng of DNA were added to 50 µL electrocompetent cells and transferred to a 0.2 cm electroporation cuvette. Electroporation parameters were: 25 µF, 200 Ω, 2500 V.
Fluorescence and growth assays
Strains were inoculated in 120 µL of MMG and transferred to 96-well plate as pre inoculum. The plate was incubated 24 h at 30 °C and 170 rpm and diluted 1:100 in a 96-well plate with MMX. Absorbance at 600 nm was measured in the Synergy™ H1 96-well Hybrid Multi-Mode Reader (BioTek® Instruments, Inc, Winooski, Vermont, USA). 96-well plates were incubated at 30 °C with continuous orbital agitation (548 cpm) in the Synergy H1 96-well plate reader. Growth (OD600) and fluorescence (RFP, exc: 535, emi: 620, gain 80) were monitored for 24 h (15 min intervals). Both parameters (fluorescence and growth) were measured in triplicate and data were expressed in arbitrary units, adjusted with the cell growth in each case.
P(3HB) accumulation assay
MM (1 g/L ammonium sulphate; xylose 15 g/L) was used to promote P(3HB) accumulation. 125 mL Erlenmeyer flasks containing 50 mL of MM were used. Initial optical density (OD600) was adjusted to 0.1 for all cultures. For the production analysis, two samples (10 mL each) were taken one at the beginning of the experiment and one after 48 h of culture (30 °C, 200 rpm). These samples were centrifuged (10,600×g, 4 °C, 10 min). Sugars were determined in the supernatant by liquid phase chromatography (HPLC) as described in “Analytical methods” section. The precipitated biomass was frozen and lyophilized to measure dry cell weight (CDW in g per liter) and to determine the composition of PHA by gas phase chromatography of propyl esters , as described in “Analytical methods” section.
10 mL of culture were harvested by centrifugation at 10,600×g and lyophilized in microtubes. In summary, samples were frozen at − 20 °C, transferred to a FreeZone Triad Cascade Benchtop Freeze Dry System (Labconco, Kansas City, Missouri, USA) and held at − 40 °C. After 2.5 h, the samples were maintained overnight at 0 °C and 0.080 mBar. After this period, the temperature was raised to 30 °C and the samples were taken from the apparatus. Dry biomass was weighed using an Adventurer Analytical Balance (Ohaus, Parsippany, New Jersey, USA) and expressed in grams per liter as cell dry weight (CDW).
Xylose concentration was determined by liquid chromatography (HPLC) as previously described . Briefly, 10 μL of each sample appropriately diluted were injected into a Ultimate 3000 HPLC (Dionex, Thermo Fisher Scientific Inc., Waltham, MA, USA) equipped with a Aminex HPX-87H Column (Bio-Rad Laboratories, Inc. Hercules, California, USA). For detection, a Refractive Index Detector Shodex RI-101 (Shodex, Munich, Germany) was used. Separation occurred at 45 °C with a H2SO4 solution (5 mM) at a flow rate of 0.6 mL/min. The standard curve was constructed using d(+)xylose solutions (Merck KGaA, Darmstadt, Germany) containing up to 3 g/L.
P(3HB) content and composition measurements
12 mg of freeze-dried cells were subjected to propanolysis  P(3HB) content was determined as described previously by Gomez  with an Agilent 7890A GC System (Agilent Technologies, Santa Clara, California, USA) equipped with an HP-5 capillary column (Agilent Technologies, Santa Clara, California, USA) after sample split (1:25). Helium (0.8 mL/min) was used as carrier gas. Benzoic acid and P(3HB) (both from Sigma-Aldrich, Saint Louis, Missouri, USA) were used as internal and external standards, respectively.
Results and discussion
Adapting BglBrick vectors to use in B. sacchari
Escherichia coli is a widely used prokaryotic host for recombinant protein production and metabolic engineering . However, since the 1990s B. sacchari, Pseudomonas putida and other Gram-negative bacteria have demonstrated enormous potential for bioproduction at an industrial scale. Despite its remarkable potential, the lack of appropriate molecular tools still limits the application of metabolic engineering or synthetic biology approaches in B. sacchari. Previously constructed broad-host-range plasmid vectors derived from IncP and IncW incompatibility groups have been shown to be able to replicate in Burkholderia species. However, most research on Burkholderia species has focused on studying multi-resistant clinical isolates [27, 35, 36], and therefore, appropriate reporter systems, inducible promoters and other synthetic biology essential tools are not available for B. sacchari.
Inducible RFP expression in B. sacchari
RFP expression was also effectively activated by pLacUV5 (Fig. 2), but in contrast to the strong inducer-dependence of pF8k-RFP, high background levels of fluorescence were observed in the strain transformed with pF5k-RFP under low induction levels (0.00625–0.0125 mM) and even in uninduced conditions (Fig. 3b). Maximum induction (2.5-fold) was reached with the addition of 0.5 mM IPTG. This result shows that while lacUV5 is functional, similar to E. coli, expression is leaky in B. sacchari, possibly due to its intrinsically higher ability to recruit RNA polymerase .
xylAB overexpression improved growth rate in B. sacchari
Increased expression of xylR further improved growth rate and P(3HB) production
Summary of the results of P(3HB) production and xylose growth rate in B. sacchari overexpressing xylR or rfp
7.15 ± 0.58
65 ± 1.98
0.20 ± 0.003
0.348 ± 0.007
5.12 ± 0.72
51.95 ± 3.01
0.156 ± 0.009
0.237 ± 0.005
8.02 ± 0.44
71.07 ± 2.46
0.252 ± 0.01
0.396 ± 0.009
5.34 ± 0.66
54 ± 2.88
0.162 ± 0.016
0.225 ± 0.006
Overexpression of xylR further improved P(3HB) accumulation
After demonstrating that xylR overexpression can improve growth on xylose, we next tested whether it can also impact P(3HB) production. P(3HB) accumulation was evaluated after 48 h in batch cultures, only for the best inducer concentration tested before (Fig. 6). As shown in Table 3, the strain expressing xylR under the control of a lacUV5 promoter induced with 6.25uM IPTG produced the highest P(3HB) titer, reaching 5.7 g/L (34% increase compared to the control strain expressing only RFP). This same strain generated the highest P(3HB) yield from xylose reported to date for B. sacchari (YP3HB/Xyl = 0.396 g/g), a 76% improvement compared to the control strain expressing RFP alone.
Prior to the results presented here, the highest reported P(3HB) yield from xylose was obtained from a wild-type B. sacchari strain which produced 0.29 gP(3HB)/gxylose with a maximum growth rate of 0.21 h−1. However, it must be mentioned that this result was obtained when B. sacchari cells were grown with a glucose to xylose ratio of 1.0 . Also emphasizing the importance of the present study, previous work from our group  determined the theoretical maximum conversion efficiency of xylose to P(3HB) using a B. sacchari metabolic model considering central metabolism, cellular biomass, and P(3HB) biosynthesis. The in silico analysis of elementary modes was performed in order to identify key metabolic modifications that would improve the xylose yield (YPHB/Xyl) of B. sacchari. This analysis determined that the theoretical maximum xylose to P(3HB) yield is 0.40 g/g  In this work, the best strain (expressing xylR under the control of pLacUV5) achieved a yield of (0.396 g/g), reaching 92.3% of the theoretical yield.
In this work we adapted two plasmids derived from BglBrick vectors allowing tunable protein expression in the non-model bacterium Burkholderia sacchari. In order to demonstrate its broad applicability and potential for bioproduction, we used these vectors to evaluate B. sacchari growth and P(3HB) accumulation in response to overexpression of all xylose isomerase pathway genes when using xylose as the sole carbon source. Overexpression of the xylose activated regulator xylR, significantly improved growth rate (0.162–0.252, 55% increase) and polymer yield (0.225–0.396, 76% increase) reaching 92.3% of the theoretical yield . In addition, the marginal improvement observed when xylFGH or xylE transporters were overexpressed strongly suggests that contrary to what has been reported for S. cerevisiae [51–53], xylose transport is not the key factor limiting xylose utilization in B. sacchari. To the best of our knowledge, this is the first report demonstrating that xylR overexpression enhances growth rate and P(3HB) production in B. sacchari.
Two plasmids for tunable gene expression in Burkholderia sacchari are presented.
xylR overexpression significantly improved growth rate and polymer yield.
The highest P(3HB) yield reported from xylose in B. sacchari was obtained (71 %CDW)
The manuscript was written with contributions from all authors. L.P.G. and C.B.O. contributed equally to the present study. All authors have read and approved the final version of the manuscript.
This work was supported by the São Paulo Research Foundation (FAPESP 2010/51989-4 and 2016/00842-0, granted to L.F.S.), National Council for Scientific and Technological Development CNPq-Brazil (454371/2014-4, granted to L.F.S.; 140321/2017-9 E.R.O.F. Ph.D. Scholarship) and Universidad Técnica de Ambato, Ecuador, through Department of Research and Development (DIDE) (Projects 0932-CU-P-2016 and 2456-CU-P-2017, granted to C.B.O.). Thanks to the National Council for Science Technology and Innovation of Ecuador (SENESCYT) for the Ph.D. fellowship granted to L.P.G.
The authors declare that they have no competing interests.
Availability of data and materials
Gene sequences used in this project are from Gen-bank (http://www.ncbi.nlm.nih.gov/). SnapGene software V3.2.1 (GSL Biotech LLC) was used to construct plasmids in silico (http://www.snapgene.com//). Escherichia coli DH10B strain is available from NEB (https://www.neb.com).
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