Tools for genetic engineering and gene expression control in
Novosphingobium aromaticivorans
and
Rhodobacter sphaeroides
Authors: Hall Ashley N. , Hall Benjamin W. , Kinney Kyle J. , Olsen Gabby G. , Banta Amy B. , Noguera Daniel R. , Donohue Timothy J. , Peters Jason M. 
Published: September 2024

Alphaproteobacteria have a variety of cellular and metabolic features that provide important insights into biological systems and enable biotechnologies. For example, some species are capable of converting plant biomass into valuable biofuels and bioproducts that have the potential to contribute to the sustainable bioeconomy. Among the Alphaproteobacteria,
Novosphingobium aromaticivorans
,
Rhodobacter sphaeroides
, and
Zymomonas mobilis
show promise as organisms that can be engineered to convert extracted plant lignin or sugars into bioproducts and biofuels. Genetic manipulation of these bacteria is needed to introduce engineered pathways and modulate expression of native genes with the goal of enhancing bioproduct output. Although recent work has expanded the genetic toolkit for
Z. mobilis
,
N. aromaticivorans
and
R. sphaeroides
still need facile, reliable approaches to deliver genetic payloads to the genome and to control gene expression. Here, we expand the platform of genetic tools for
N. aromaticivorans
and
R. sphaeroides
to address these issues. We demonstrate that Tn
7
transposition is an effective approach for introducing engineered DNA into the chromosome of
N. aromaticivorans
and
R. sphaeroides
. We screen a synthetic promoter library to identify isopropyl β-D-1-thiogalactopyranoside-inducible promoters with regulated activity in both organisms (up to ~15-fold induction in
N. aromaticivorans
and ~5-fold induction in
R. sphaeroides
). Combining Tn
7
integration with promoters from our library, we establish CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) interference systems for
N. aromaticivorans
and
R. sphaeroides
(up to ~10-fold knockdown in
N. aromaticivorans
and
R. sphaeroides
) that can target essential genes and modulate engineered pathways. We anticipate that these systems will greatly facilitate both genetic engineering and gene function discovery efforts in these species and other Alphaproteobacteria.

IMPORTANCE

It is important to increase our understanding of the microbial world to improve health, agriculture, the environment, and biotechnology. For example, building a sustainable bioeconomy depends on the efficient conversion of plant material to valuable biofuels and bioproducts by microbes. One limitation in this conversion process is that microbes with otherwise promising properties for conversion are challenging to genetically engineer. Here we report genetic tools for
Novosphingobium aromaticivorans
and
Rhodobacter sphaeroides
that add to the burgeoning set of tools available for genome engineering and gene expression in Alphaproteobacteria. Our approaches allow straightforward insertion of engineered pathways into the
N. aromaticivorans
or
R. sphaeroides
genome and control of gene expression by inducing genes with synthetic promoters or repressing genes using CRISPR interference. These tools can be used in future work to gain additional insight into these and other Alphaproteobacteria and to aid in optimizing yield of biofuels and bioproducts.