Combinatorial metabolic engineering of Pseudomonas putida KT2440 for efficient mineralization of 1,2,3-trichloropropane

Ting Gong,Xiaoqing Xu,Huilei Yu,Ruihua Liu,Ping Xu,Weixia Gao,You Che,Chao Yang,Fengjie Zhao,Jingnan Liang,Cunjiang Song

doi:10.1038/s41598-017-07435-x

Abstract

An industrial waste, 1,2,3-trichloropropane (TCP), is toxic and extremely recalcitrant to biodegradation. To date, no natural TCP degraders able to mineralize TCP aerobically have been isolated. In this work, we engineered a biosafety Pseudomonas putida strain KT2440 for aerobic mineralization of TCP by implantation of a synthetic biodegradation pathway into the chromosome and further improved TCP mineralization using combinatorial engineering strategies. Initially, a synthetic pathway composed of haloalkane dehalogenase, haloalcohol dehalogenase and epoxide hydrolase was functionally assembled for the conversion of TCP into glycerol in P. putida KT2440. Then, the growth lag-phase of using glycerol as a growth precursor was eliminated by deleting the glpR gene, significantly enhancing the flux of carbon through the pathway. Subsequently, we improved the oxygen sequestering capacity of this strain through the heterologous expression of Vitreoscilla hemoglobin, which makes this strain able to mineralize TCP under oxygen-limited conditions. Lastly, we further improved intracellular energy charge (ATP/ADP ratio) and reducing power (NADPH/NADP+ ratio) by deleting flagella-related genes in the genome of P. putida KT2440. The resulting strain (named KTU-TGVF) could efficiently utilize TCP as the sole source of carbon for growth. Degradation studies in a bioreactor highlight the value of this engineered strain for TCP bioremediation.

Highlights

Synthetic biology has become a powerful tool to construct complete heterologous metabolic pathways in a host cell by functional assembly of various enzymes from different organisms[1]
A synthetic pathway for aerobic mineralization of TCP comprising DhaA from Rhodococcus rhodochrous NCIMB 13064, HheC from Agrobacterium radiobacter AD1, and EchA from Agrobacterium radiobacter AD1 was assembled in P. putida KT2440 (Fig. 1)
The concentration of TCP did not change and no growth was observed when inoculated with KTU. These results indicated that TCP could be converted to GLY by the heterologous pathway assembled in P. putida KT2440 (Fig. 1)

Summary

Introduction

Synthetic biology has become a powerful tool to construct complete heterologous metabolic pathways in a host cell by functional assembly of various enzymes from different organisms[1]. Synthetic biology tools are not widely applied for engineering the organics catabolic pathways in microorganisms so far, they have enormous potential for rational tuning of pathways for efficient degradation of environmental pollutants. Metabolic engineering has opened up new avenues for the evolution of efficient degradation pathways[6], which allows the construction of recombinant microorganisms with the capability to degrade TCP under aerobic conditions. A synthetic biodegradation pathway capable of aerobic biotransformation of TCP into harmless GLY was assembled by the heterologous expression of DhaA, HheC and EchA in Escherichia coli[12, 13]. The engineered strain needs to be intensively researched with regard to its biosafety prior to its application for in situ bioremediation due to legislative barriers on the field release of genetically engineered microorganisms

Methods

Results

Conclusion