Autotrophic lactate production from H2\u2009+\u2009CO2 using recombinant and fluorescent FAST-tagged Acetobacterium woodii strains

Alexander Mook,Frank R Bengelsdorf,Nigel P Minton,Peter Dürre,Jonathan P Baker,Matthias H Beck

doi:10.1007/s00253-022-11770-z

Abstract

Lactate has various uses as industrial platform chemical, poly-lactic acid precursor or feedstock for anaerobic co-cultivations. The aim of this study was to construct and characterise Acetobacterium woodii strains capable of autotrophic lactate production. Therefore, the lctBCD genes, encoding the native Lct dehydrogenase complex, responsible for lactate consumption, were knocked out. Subsequently, a gene encoding a d-lactate dehydrogenase (LDHD) originating from Leuconostoc mesenteroides was expressed in A. woodii, either under the control of the anhydrotetracycline-inducible promoter Ptet or under the lactose-inducible promoter PbgaL. Moreover, LDHD was N-terminally fused to the oxygen-independent fluorescence-activating and absorption-shifting tag (FAST) and expressed in respective A. woodii strains. Cells that produced the LDHD fusion protein were capable of lactate production of up to 18.8 mM in autotrophic batch experiments using H2 + CO2 as energy and carbon source. Furthermore, cells showed a clear and bright fluorescence during exponential growth, as well as in the stationary phase after induction, mediated by the N-terminal FAST. Flow cytometry at the single-cell level revealed phenotypic heterogeneities for cells expressing the FAST-tagged LDHD fusion protein. This study shows that FAST provides a new reporter tool to quickly analyze gene expression over the course of growth experiments of A. woodii. Consequently, fluorescence-based reporters allow for faster and more targeted optimization of production strains.Key points•Autotrophic lactate production was achieved with A. woodii.•FAST functions as fluorescent marker protein in A. woodii.•Fluorescence measurements on single-cell level revealed population heterogeneity.

Highlights

IntroductionThe recent and ever-developing pursuit of a clean and sustainable industry raises the need for novel, especially biological, processes that use gaseous waste streams to generateAlexander Mook and Matthias H
After recombinant ldhD gene expression was induced by ATc, the latter two strains produced up to 10 mM lactate under autotrophic growth conditions
A. woodii Ptet_ldhDCI reached a peak acetate concentration of only 102 mM. Both A. woodii [Ptet_ldhD] and A. woodii Ptet_ldhDCI were able to produce about 10 mM lactate, despite differences in growth (Fig. 1C)

Summary

Introduction

The recent and ever-developing pursuit of a clean and sustainable industry raises the need for novel, especially biological, processes that use gaseous waste streams to generateAlexander Mook and Matthias H. The recent and ever-developing pursuit of a clean and sustainable industry raises the need for novel, especially biological, processes that use gaseous waste streams to generate. Autotrophic fermentations employing acetogens that grow by using mixtures of CO2, CO and H2 offer a future-proof biological process, converting industrial waste gas streams to a variety of organic acids and alcohols (Bengelsdorf and Dürre 2017). CO2-based processes are getting more and more attractive and economically feasible, due to efforts to increase the costs of CO2 emission and reduce greenhouse gas emissions. One potential chemical that can be produced from C O2 is lactate (Eş et al 2018). There are recent approaches to use lactate as feedstock in (synthetic) co-cultures to produce value-added

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