Elsevier

Available online 29 July 2021

New Biotechnology

Highlights

Ineffective electroporation of Cupriavidus necator H16 with pBBR1MCS-2 is explained.

Minimal, modular vectors (pCAT) with broad host range replication machinery were constructed.

pCAT vectors transform C. nector via electroporation with high efficiency.

Genes cloned in pCAT vectors are stably propagated.

pCAT one-pot multi-fragment assemblies can be electroporated directly into C. necator.

Abstract

Cupriavidus necator H16 is a chemolithoautotroph with a range of industrial biotechnological applications. Advanced metabolic engineering in the bacterium, however, is impeded by low transformation efficiency, making it difficult to introduce and screen new genetic functions rapidly. This study systematically characterized the broad host range plasmids pBHR1, pBBR1MCS-2 and pKT230 used frequently for C. necator engineering. Kanamycin resistance cassette (KanR) and a truncated sequence of the replication origin (Rep) are contributing factors to C. necator low electroporation transformation efficiency. Consequently, a series of modular minimal plasmids, named pCAT, was constructed. pCAT vectors transform C. necator H16 with a > 3000-fold higher efficiency (up to 107 CFU/μg DNA) compared to control plasmids. Further, pCAT vectors are highly stable, expressing reporter proteins over several days of serial cultivation in the absence of selection pressure. Finally, they can be assembled rapidly from PCR or synthesized DNA fragments, and restriction-ligation reactions can be efficiently electroporated directly into C. necator, circumventing the requirement to use Escherichia coli for plasmid maintenance or propagation. This study demonstrates that an understanding of the behaviour of the constituent parts of plasmids in a host is key to efficient propagation of genetic information, while offering new methods for engineering a bacterium with desirable industrial biotechnological features.

Abbreviations

pCAT

minimal modular broad host range plasmid characterized for propagation in Cupriavidus necator

KanR

kanamycin resistance cassette

APH (3’)-Ia

aminoglycoside O-phosphotransferase class I subtype ‘a’

APH (3’)-IIa

aminoglycoside O-phosphotransferase class II subtype ‘a’

CmR

chloramphenicol resistant cassette

EryR

erythromycin resistance cassette

TcR

tetracycline resistance cassette

AmpR

ampicillin resistant cassette

CarbR

carbenicillin resistance cassette

MCS

multiple cloning sites

OriV

vegetative origin of replication

mRFP1

mcherry red fluorescent protein

eGFP

enhanced green fluorescent protein

pSEVA

Standard European Vector Architecture

SOC

Super optimal broth with catabolite repression fructose or glucose

Keywords

Cupriavidus necator

Electroporation

Plasmid assembly

Biopart characterization

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© 2021 Published by Elsevier B.V.



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