A Nitrate-Blind P. putida Strain Boosts PHA Production in a Synthetic Mixed Culture.

Karina Hobmeier,Hannes Löwe,Stephan Liefeldt,Andreas Kremling,Katharina Pflüger-Grau

doi:10.3389/fbioe.2020.00486

Karina Hobmeier, Hannes Löwe + Show 3 more

Open Access

https://doi.org/10.3389/fbioe.2020.00486

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Abstract

One of the major challenges for the present and future generations is to find suitable substitutes for the fossil resources we rely on today. In this context, cyanobacterial carbohydrates have been discussed as an emerging renewable feedstock in industrial biotechnology for the production of fuels and chemicals. Based on this, we recently presented a synthetic bacterial co-culture for the production of medium-chain-length polyhydroxyalkanoates (PHAs) from CO2. This co-cultivation system is composed of two partner strains: Synechococcus elongatus cscB which fixes CO2, converts it to sucrose and exports it into the culture supernatant, and a Pseudomonas putida strain that metabolizes this sugar and accumulates PHAs in the cytoplasm. However, these biopolymers are preferably accumulated under conditions of nitrogen limitation, a situation difficult to achieve in a co-culture as the other partner, at best, should not perceive any limitation. In this article, we will present an approach to overcome this dilemma by uncoupling the PHA production from the presence of nitrate in the medium. This is achieved by the construction of a P. putida strain that is no longer able to grow with nitrate as nitrogen source -is thus nitrate blind, and able to grow with sucrose as carbon source. The deletion of the nasT gene encoding the response regulator of the NasS/NasT two-component system resulted in such a strain that has lost the ability use nitrate, but growth with ammonium was not affected. Subsequently, the nasT deletion was implemented in P. putida cscRABY, an efficient sucrose consuming strain. This genetic engineering approach introduced an artificial unilateral nitrogen limitation in the co-cultivation process, and the amount of PHA produced from light and CO2 was 8.8 fold increased to 14.8% of its CDW compared to the nitrate consuming reference strain. This nitrate blind strain, P. putidaΔnasT attTn7:cscRABY, is not only a valuable partner in the co-cultivation but additionally enables the use of other nitrate containing substrates for medium-chain-length PHA production, like for example waste-water.

Highlights

In times of global warming, extreme weather conditions, and a growing world population, it is mandatory to dedicate arable land to food production and not “waste” it for energy formation or feedstock production for biotechnology
A clean deletion of nasT was introduced into P. putida EM178, a prophage free derivative of P. putida KT2440 by I-SceI aided double homologous recombination (Martínez-García and de Lorenzo, 2011)
We reported that deletion of the nasT gene encoding the response regulator of the NasS/NasT two component system resulted in a strain insensitive to the presence of nitrate and unable to grow with nitrate as nitrogen source

Summary

Introduction

In times of global warming, extreme weather conditions, and a growing world population, it is mandatory to dedicate arable land to food production and not “waste” it for energy formation or feedstock production for biotechnology. A number of studies were published recently, that employed a genetically engineered Synechococcus elongatus PCC7942 strain in a functional mixed culture (Hays and Ducat, 2015; Smith and Francis, 2016; Li et al, 2017; Löwe et al, 2017; Weiss et al, 2017). We recently set up a co-cultivation for the production of polyhydroxyalkanoates (PHA) with S. elongatus PCC7942 cscB and the genetically engineered strain P. putida:mini-Tn5(cscAB), capable of metabolizing sucrose (Löwe et al, 2017) With this mixed culture approach a production rate of PHA of around 23.8 mg L−1 d−1 was reached under nitrogen limiting conditions. P. putida is a very suitable partner for co-cultivations as it combines various traits, including its genetic tractability and its general stress resistance (Nikel et al, 2016), which is of great importance when grown in the non-optimal environment of the photobioreactor with elevated salt concentrations

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