Elsevier

Metabolic Engineering

Volume 14, Issue 4, July 2012, Pages 317-324
Metabolic Engineering

Production and characterization of poly(3-hydroxypropionate-co-4-hydroxybutyrate) with fully controllable structures by recombinant Escherichia coli containing an engineered pathway

https://doi.org/10.1016/j.ymben.2012.04.003Get rights and content

Abstract

Copolyesters of 3-hydroxypropionate (3HP) and 4-hydroxybutyrate (4HB), abbreviated as P(3HP-co-4HB), was synthesized by Escherichia coli harboring a synthetic pathway consisting of five heterologous genes including orfZ encoding 4-hydroxybutyrate-coenzyme A transferase from Clostridium kluyveri, pcs’ encoding the ACS domain of tri-functional propionyl-CoA ligase (PCS) from Chloroflexus aurantiacus, dhaT and aldD encoding dehydratase and aldehyde dehydrogenase from Pseudomonas putida KT2442, and phaC1 encoding PHA synthase from Ralstonia eutropha. When grown on mixtures of 1,3-propanediol (PDO) and 1,4-butanediol (BDO), compositions of 4HB in microbial P(3HP-co-4HB) were controllable ranging from 12 mol% to 82 mol% depending on PDO/BDO ratios. Nuclear magnetic resonance (NMR) spectra clearly indicated the polymers were random copolymers of 3HP and 4HB. Their mechanical and thermal properties showed obvious changes depending on the monomer ratios. Morphologically, P(3HP-co-4HB) films only became fully transparent when monomer 4HB content was around 67 mol%. For the first time, P(3HP-co-4HB) with adjustable monomer ratios were produced and characterized.

Highlights

► An engineering pathway in recombinant Escherichia coli was constructed for producing novel biomaterials P(3HP-co-4HB). ► The pathway allows 3HP/4HB ratios in P(3HP-co-4HB) to be controllable. ► Flexible mechanical and thermal properties of P(3HP-co-4HB) were obtained.

Introduction

Metabolic engineering of microorganisms has been exploited as a powerful approach for enhanced production of chemicals, polymeric materials and biofuels from renewable feedstocks (Carole et al., 2004, Dugar and Stephanopoulos, 2011, Krivoruchko et al., 2011, Lee et al., 2011a, Yim et al., 2011). Polyhydroxyalkanoates (PHA), a family of biodegradable and biocompatible polyesters produced by a variety of microorganisms, have been extensively studied using metabolic engineering methods both for improving PHA production and for widening the range of PHA diversity (Chen and Patel, 2011, Steinbüchel and Fuchtenbusch, 1998, Verlinden et al., 2007). Microbial metabolic engineering has indeed allowed PHA to become not just composition controllable random copolymers, but also be turned into PHA homopolymers and block copolymers as well as chiral monomers that are useful for the fine chemicals and pharmaceutical industries (Chen, 2009, Chen and Wu, 2005, Cheng et al., 2006, Gao et al., 2011, Hu et al., 2011, Lee et al., 2011b, Liu et al., 2011, Martin and Williams, 2003, Tan et al., 2011).

The PHA family shows diverse monomers and compositions as well as fine structures (Chung et al., 2011, Ishida et al., 2001, Saito et al., 1996, Steinbüchel and Valentin, 1995). Short-chain-length (SCL) PHA consists of monomers with carbon chain length from C3 to C5 including 3-hydroxypropionate (3HP), 3-hydroxybutyrate (3HB), 4-hydroxybutyrate (4HB) and 3-hydroxyvalerate (3HV) (Hu et al., 2011, Sudesh et al., 2000), while medium-chain-length (MCL) PHA contain monomers from C6 to C14 (Witholt and Kessler, 1999). Copolymers consisting of SCL- and MCL monomers are attracting many attentions due to their flexible mechanical properties (Chen et al., 2004).

Recently, microbial copolyesters containing 3HP are becoming increasingly interesting due to the ultrahigh strength brought about by 3HP, these include P(3HB-co-3HP), P(3HP-co-3HB-co-3HH-co-3HO), P(4HB-co-3HP-co-Lactate), P(4HB-co-3HP-co-2HP), P(3HB-co-3HP-4HB-co-Lactate), and P(3HB-co-3HP-co-4HB-co-2HP) (Andreessen and Steinbüchel, 2010). We recently reported the biosynthesis of P(3HP-co-4HB) containing less than 2 mol% 4HB (Zhou et al., 2011). However, we were unable to manipulate the bacterium to control the 4HB compositions in the P(3HP-co-4HB) copolymer for property optimization.

Several key enzymes were considered as important for making P(3HP-co-4HB) copolymers with flexible 4HB content: propionyl-CoA synthetase (PCS’) from the 3-hydroxypropionate cycle of phototrophic green non-sulfur eubacterium Chloroflexus aurantiacus is very likely to convert 3HP to 3HP-CoA although PCS’ has very lower activity for converting 4HB to 4HB-CoA (Alber and Fuchs, 2002, Herter et al., 2002, Hugler et al., 2002). 4HB-Coenzyme A transferase gene orfZ from Clostridium kluyveri was found able to turn 4HB into 4HB-CoA effectively (Hein et al., 1997, Song et al., 1999, Zhang et al., 2009). Genes dhaT and aldD were proven functional to synthesize 1,4-butanediol (BDO) or/and 1,3-propanediol (PDO) to 4HB or/and 3HP, the enzyme encoded by dhaT was mostly active with substrates containing two primary alcohol groups separated by one or two carbon atoms such as 1,3-propanediol or 1,4-butanediol, and 3HP or/and 4HB yield were affected by expression levels of these two genes (Zhang et al., 2009). Promoter of PHA synthesis genes phaCAB operon from Ralstonia eutropha (PRe) was demonstrated to be more active than lac promoter or T7 promoter transcriptionally in E. coli (Studier and Moffatt, 1986, Zhou et al., 2011). Finally, PHA synthase PhaC1 of R. eutropha has sufficient activity for polymerizing SCL PHA monomers (Hu et al., 2011).

With the understanding of key enzymes for making P(3HP-co-4HB) copolymers consisting of flexible 4HB content, this study aimed at constructing pathways in E. coli for production of P(3HP-co-4HB) consisting of a series 4HB ratios, characterization of these copolymers should allow understanding of their structure and property relationship for our material application purposes (Andreessen and Steinbüchel, 2010).

Section snippets

Microorganisms, plasmids and genetic methods

Escherichia coli Trans1-T1, as the fastest growing and competent strain currently available, was used as the host for plasmids construction and as a vector donor in conjugation. E. coli S17-1 was used for P(3HP-co-4HB) accumulation (Simon et al., 1983, Zhou et al., 2011). All plasmids and bacterial strains used in this study are listed in Table 1. Primers used for PCR and for constructions of new plasmids are synthesized by AuGCT Biotech (Beijing, China).

Plasmid pZQ03-orfZ was pZQ03 derivatives

Construction of P(3HP-co-4HB) engineering pathway

Natural bacteria are unable to produce 3-hydroxyproionate (3HP) and 4-hydroxybutyrate (4HB) as building blocks for PHA synthase to make the unnatural biopolyester P(3HP-co-4HB). However, precusors of 3HP and 4HB can come from 1,3-propanediol (PDO) and 1,4-butanediol (BDO), respectively. While PDO and BDO can be, respectively biosynthesized from glucose (Celinska, 2010, Yim et al., 2011). It thus becomes possible to establish an engineering pathway for production of P(3HP-co-4HB). In this study,

Discussion

P3HP was demonstrated as the PHA with the best mechanical properties so far reported (Zhou et al., 2011). While it was well known that 4HB enhanced elasticity of PHA (Saito et al., 1996). Thus, it looks like that a copolymer of P(3HP-co-4HB) should produce desirable properties for some applications. However, not a natural pathway was even established to produce this copolymer.

This study successfully constructed an engineering pathway in recombinant Escherichia coli consisting of enzymes

Conclusion

Escherichia coli expressing a synthetic pathway consisting of five heterologous genes including orfZ from Clostridium kluyveri, pcs’ from Chloroflexus aurantiacus, dhaT and aldD from Pseudomonas putida KT2442, and phaC1 from Ralstonia eutropha, was able to synthesize copolyesters of P(3HP-co-4HB) when grown on 1,3-propanediol (PDO) and/or 1,4-butanediol (BDO). A series of P(3HP-co-4HB) with 4HB ratios ranging from 0 mol% to 100 mol% were produced depending on PDO/BDO ratios. Their mechanical and

Acknowledgments

We are grateful for the generous donation of plasmid pBHR68 from Professor Alexander Steinbüchel of the University of Münster in Germany. This research was supported by the State Basic Science Foundation 973 (Grant No. 2012CB725201, Grant No. 2012CB725204, Grant No. 2012CB725200 and 2011CBA00807) and a Grant from National Natural Science Foundation of China (Grant No. 31170099).

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