Malonate degradation in Acinetobacter baylyi ADP1: operon organization and regulation by MdcR

ABSTRACTOne goal of synthetic biology is to improve the efficiency and predictability of living cells by removing extraneous genes from their genomes. We demonstrate improved methods for engineering the genome of the metabolically versatile and naturally transformable bacterium Acinetobacter baylyi ADP1 and apply them to a genome streamlining project. In Golden Transformation, linear DNA fragments constructed by Golden Gate Assembly are directly added to cells to create targeted deletions, edits, or additions to the chromosome. We tested the dispensability of 55 regions of the ADP1 chromosome using Golden Transformation. The 19 successful multiple-gene deletions ranged in size from 21 to 183 kilobases and collectively accounted for 24.6% of its genome. Deletion success could only be partially predicted on the basis of a single-gene knockout strain collection and a new Tn-Seq experiment. We further show that ADP1’s native CRISPR/Cas locus is active and can be retargeted using Golden Transformation. We reprogrammed it to create a CRISPR-Lock, which validates that a gene has been successfully removed from the chromosome and prevents it from being reacquired. These methods can be used together to implement combinatorial routes to further genome streamlining and for more rapid and assured metabolic engineering of this versatile chassis organism.

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Rapid and assured genetic engineering methods applied to Acinetobacter baylyi ADP1 genome streamlining

Nucleic Acids Research ◽

10.1093/nar/gkaa204 ◽

2020 ◽

Vol 48 (8) ◽

pp. 4585-4600

Author(s):

Gabriel A Suárez ◽

Kyle R Dugan ◽

Brian A Renda ◽

Sean P Leonard ◽

Lakshmi Suryateja Gangavarapu ◽

...

Keyword(s):

Gene Deletion ◽

Single Gene ◽

Acinetobacter Baylyi ◽

Gene Deletions ◽

Multiple Gene ◽

Transposon Insertion ◽

A Genome ◽

Acinetobacter Baylyi Adp1 ◽

Transposon Insertion Sequencing ◽

Improved Methods

Abstract One goal of synthetic biology is to improve the efficiency and predictability of living cells by removing extraneous genes from their genomes. We demonstrate improved methods for engineering the genome of the metabolically versatile and naturally transformable bacterium Acinetobacter baylyi ADP1 and apply them to a genome streamlining project. In Golden Transformation, linear DNA fragments constructed by Golden Gate Assembly are directly added to cells to create targeted deletions, edits, or additions to the chromosome. We tested the dispensability of 55 regions of the ADP1 chromosome using Golden Transformation. The 18 successful multiple-gene deletions ranged in size from 21 to 183 kb and collectively accounted for 23.4% of its genome. The success of each multiple-gene deletion attempt could only be partially predicted on the basis of an existing collection of viable ADP1 single-gene deletion strains and a new transposon insertion sequencing (Tn-Seq) dataset that we generated. We further show that ADP1’s native CRISPR/Cas locus is active and can be retargeted using Golden Transformation. We reprogrammed it to create a CRISPR-Lock, which validates that a gene has been successfully removed from the chromosome and prevents it from being reacquired. These methods can be used together to implement combinatorial routes to further genome streamlining and for more rapid and assured metabolic engineering of this versatile chassis organism.

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Development of a genetic toolset for the highly engineerable and metabolically versatile Acinetobacter baylyi ADP1

Nucleic Acids Research ◽

10.1093/nar/gkaa167 ◽

2020 ◽

Vol 48 (9) ◽

pp. 5169-5182

Author(s):

Bradley W Biggs ◽

Stacy R Bedore ◽

Erika Arvay ◽

Shu Huang ◽

Harshith Subramanian ◽

...

Keyword(s):

Synthetic Biology ◽

Carbon Sources ◽

Primary Objective ◽

Single Step ◽

Chromosomal Protein ◽

Acinetobacter Baylyi ◽

Catabolic Repression ◽

Chemical Manufacturing ◽

Promoter Library ◽

Acinetobacter Baylyi Adp1

Abstract One primary objective of synthetic biology is to improve the sustainability of chemical manufacturing. Naturally occurring biological systems can utilize a variety of carbon sources, including waste streams that pose challenges to traditional chemical processing, such as lignin biomass, providing opportunity for remediation and valorization of these materials. Success, however, depends on identifying micro-organisms that are both metabolically versatile and engineerable. Identifying organisms with this combination of traits has been a historic hindrance. Here, we leverage the facile genetics of the metabolically versatile bacterium Acinetobacter baylyi ADP1 to create easy and rapid molecular cloning workflows, including a Cas9-based single-step marker-less and scar-less genomic integration method. In addition, we create a promoter library, ribosomal binding site (RBS) variants and test an unprecedented number of rationally integrated bacterial chromosomal protein expression sites and variants. At last, we demonstrate the utility of these tools by examining ADP1’s catabolic repression regulation, creating a strain with improved potential for lignin bioprocessing. Taken together, this work highlights ADP1 as an ideal host for a variety of sustainability and synthetic biology applications.

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