Engineering of Ralstonia eutropha for the production of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) from glucoseJournal of Biotechnology


Ying-Zi Zhang, Gui-Ming Liu, Wei-Qi Weng, Jiu-Yuan Ding, Shuang-Jiang Liu
Applied Microbiology and Biotechnology / Biotechnology


Journal of Biotechnology 195 (2015) 82–88

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Journal of Biotechnology j ourna l ho me pa ge: www.elsev ier .com/ locate / jb io tec

Engineering of Ralstonia eutropha for the product poly(3- ) f

Ying-Zi Z Din a State Key Lab Beijin b CAS Key Labo inese a r t i c l

Article history:

Received 6 Oc

Received in re

Accepted 6 De

Available onlin


Ralstonia eutro

Poly(3-hydrox hydroxyvalera (PHBV)

Propionyl-CoA 2-Methylcitric acid cycle

Methylmalonyl-CoA e-coenta n. In ethylm and ions lly en tion o 1. Introdu

Poly(3-h biodegrada commercia 1992). Ferm propionate

Lee, 1999). with its hig main factor from being

Efforts hav processes t purpose, se enterica we carbon sou 2014; Wan

The 3-h flexibility, ∗ Correspon ences, Beichen

Tel.: +86 10 64

E-mail add http://dx.doi.o 0168-1656/© ction ydroxybutyrate-co-3-hydroxyvalerate) (PHBV) are ble polyesters of industrial applications and have been lized with the name Biopol® (Byrom, 1987; Luzier, entative production of PHBV relies on the addition of as a precursor for propionyl-CoA synthesis (Choi and

The toxicity of propionate to bacterial growth, along h cost (Aldor et al., 2002; Horswill et al., 2001), are the s that prevent propionate-dependent PHBV production a cost-effective process (Aldor and Keasling, 2001). e been made to establish alternative fermentative hat are independent of propionate addition. For this veral bacteria such as Escherichia coli and Salmonella re engineered for PHBV synthesis from unrelated rces (Aldor et al., 2002; Chen et al., 2011; Yang et al., g et al., 2014). ydroxyvalerate (3HV) fraction contributes to the elasticity and melting temperature as well as the ding authors at: Institute of Microbiology, Chinese Academy of Sci-Xilu No. 1, Chaoyang District, Beijing 100101, PR China. 807423; fax: +86 10 64807421. resses: (J.-Y. Ding), (S.-J. Liu). processing properties of PHBV. When the molar ratio of 3HV to 3-hydroxybutyrate (3HB) is approximately 20 mol%, PHBV has excellent strength and flexibility (Luzier, 1992) and can be easily processed for nanocomposites and medical applications (Zinn et al., 2001; Yu and Qin, 2014). Studies have showed that the supply of propionyl-CoA in cells is the key factor governing the 3HV fraction in PHBV (Aldor et al., 2002). Haloferax mediterranei is a natural PHBV producer (Don et al., 2006). Recent studies showed that four metabolic pathways (citramalate/2oxobutyrate, aspartate/2-oxobutryate, methylmalonyl-CoA, and 3hydroxypropionate, respectively) were responsible for propionylCoA synthesis in H. mediterranei (Han et al., 2013). Meanwhile, various strategies were applied to increase the propionyl-CoA supply, including the establishment of a citramalate pathway (Yang et al., 2014), manipulation of branched amino acids (e.g., threonine, isoleucine) pathways (Steinbüchel and Pieper, 1992; Chen et al., 2011), and engineering of a methylmalonyl-CoA pathway (Aldor et al., 2002). Thus far, the construction of bacterial PHBV producers with high productivity and better fermentation performance with low production costs remain a great interest to industry and scientists.

Ralstonia eutropha strain H16 is a historical and model organism for the investigation of PHB synthesis (Doi et al., 1989; Madison and

Huisman, 1999; Peoples and Sinskey, 1989). Currently, derivatives of strain H16 are the main workhorse for commercial production rg/10.1016/j.jbiotec.2014.12.014 2014 Elsevier B.V. All rights reserved.hydroxybutyrate-co-3-hydroxyvalerate hanga,b, Gui-Ming Liub, Wei-Qi Wengb, Jiu-Yuan oratory of Microbial Resources, Institute of Microbiology, Chinese Academy of Sciences, ratory of Microbial Physiology and Metabolic Engineering, Institute of Microbiology, Ch e i n f o tober 2014 vised form 4 December 2014 cember 2014 e 23 December 2014 pha ybutyrate-co-3te) a b s t r a c t

Production of poly(3-hydroxybutyrat the addition of propionate during ferm affecting the cost of PHBV productio the methylcitric acid cycle and the m prpC2 genes did not affect cell growth high amounts of PHBVs with 3HV fract 7.5-L fermenter showed that genetica which 68.6% were PHBV with 3HV frac of commercialization in the future.ion of rom glucose gb,∗, Shuang-Jiang Liua,∗ g 100101, PR China

Academy of Sciences, Beijing 100101, PR China 3-hydroxyvalerate) (PHBV) with Ralstonia eutropha relies on tion, and propionate consumption is one of the major factors this study, 7 strains were obtained by genetic manipulating alonyl-CoA pathway in R. eutropha. Disruption of prpC1 and

PHBV accumulation. All 7 strains were able to accumulation of 0.41–29.1 mol% during cultivation in flasks. Fermentation in gineered Rem-8 was able to yield biomass of 132.8 CDW g/L, of f 26.0 mol% in the biopolymer, indicating promising potentials © 2014 Elsevier B.V. All rights reserved.

Y.-Z. Zhang et al. / Journal of Biotechnology 195 (2015) 82–88 83

Table 1

Bacterial strains, plasmids, and primers used in this study.

Strains and plasmids Relevant characteristics Sources or references


Escherichia c



Ralstonia eut

Rem-1 (prev



Rem-4 f


Rem-6 f


Rem-8 or pZM

Plasmids pMD19-T pBBR1MCS pJQ200mp18 pZP4 pha pZMwf m W3 pJQ200mp18 rpC1 pJQ200mp18 rpC2



Pcab-4r muyayf muyayr prpC1df prpC1dr prpC2df prpC2dr of PHB and

Steinbüche producers et al., 2002 the advanta study, we ge 1 (formerly eutropha H1 were obtain cultivation 2. Materia 2.1. Bacteri growth cond

All strain this study a derived from assimilation

E. coli S

Bertani (LB

NaCl), supp (100 g/mL broth or 80 eutropha str with glucos 2.2. Produc

Producti evaluated i (Berezina, 2 30 ◦C and w ment so ev

SA). of m lled 00 m the oli DH5 80 LacZ M15, deoR, recA1, endA1, hsdR17

Wild type, F-, -,rphF-mcrA, lacZM15, lacX74, Tra+ ropha H16 Wild type, DSM 428 iously 65-7) Ralstonia eutropha H16 glucose mutant