Biology subjects

Nielsen, D. R.

Publications and source records attributed to Nielsen, D. R..

4 recordsLinked to original sources

bioRxiv2024

Highly Iterated Palindrome 1 (HIP1) sequence improves Synechococcus sp. PCC 7002 transformation efficiencies in a homology- and methylation-dependent manner

The ability to precisely control cyanobacterial metabolism first requires the ability to efficiently deliver engineered DNA constructs. Here, we investigate how natural transformation efficiencies in Synechococcus sp. PCC 7002 can be greatly improved by leveraging the native and abundant cyanobacterial Highly Iterated Palindrome 1 (HIP1) sequence. While including at least one homologous HIP1 site within the homology arms of an integrating plasmid increased integration efficiency by up to 7-fold, methylation of those sites by HIP1 methyltransferase (encoded by slr0214 from Synechococcus sp. PCC 6803) boosted this to greater than a 100-fold improvement overall. Non-homologous HIP1 sites also improved transformation efficiencies of both integrating and replicating episomal plasmids (by up to 60- and 9-fold, respectively), but only when methylated. The collective data further reveal that HIP1 does not function as part of a native restriction enzyme system in PCC 7002, but rather may improve transformation efficiency via two complementary mechanisms: i) altering DNA binding/uptake/processing prior to homologous recombination, and ii) increasing the efficiency of homologous recombination in a manner reminiscent of a crossover hotspot instigator (Chi) site. Future studies are needed, however, to more clearly elucidate the specific role of HIP1 during natural transformation of cyanobacteria.

synthetic biology↗

bioRxiv2024

Delivery of novel replicating vectors to Synechococcus sp. PCC 7002 via natural transformation of plasmid multimers

In most cyanobacteria, genetic engineering efforts currently rely upon chromosomal integration; a time-consuming process due to their polyploid nature. To enhance strain construction, here we develop and characterize two novel replicating plasmids for use in Synechococcus sp. PCC 7002. Following an initial screen of plasmids comprising seven different origins of replication, two were found capable of replication: one based on the WVO1 broad host range plasmid and the other a shuttle vector derived from pCB2.4 from Synechocystis sp. PCC 6803. These were then used to construct a set of new replicating plasmids, which were shown to be both co-transformable and stably maintained in PCC 7002 at copy numbers between 0.6-1.4 and 7-16, respectively. Lastly, we demonstrate the importance of using multimeric plasmids during natural transformation of PCC 7002, with higher order multimers providing a 30-fold increase in transformation efficiency relative to monomeric plasmids. Useful considerations and methods for enhancing multimer content in plasmid samples are also presented.

synthetic biology↗

bioRxiv2024

In situ, High-Resolution Quantification of CO2 Uptake Rates via Automated Off-Gas Analysis Illuminates Carbon Uptake Dynamics in Cyanobacterial Cultures

Quantification of CO2 fixation rates by cyanobacteria is vital to determining their potential as industrial strains in a circular bioeconomy. Currently, however, CO2 fixation rates are most often determined through indirect and/or low-resolution methods, resulting in an incomplete picture of both dynamic behaviors and total carbon fixing potential. To address this, we developed a novel, low-cost system for in situ off-gas analysis which supports the automated acquisition of high-resolution and CO2 uptake rates from cyanobacterial cultures. Carbon fixation data obtained via this system was independently verified by elemental analysis of cultivated biomass. Using Synechococcus sp. PCC 7002 and Synechocystis sp. PCC 6803, we demonstrate that phototrophic CO2 uptake rates accelerate linearly to a maximum before then decaying monotonically to cessation by stationary phase. Furthermore, consistent with the expected stoichiometry, we found that strong correlations exist between both the rates and total levels of cell growth and carbon fixation. This system simultaneously provides a high-resolution growth curve, accurate carbon fixation rates, as well as the total amount of carbon fixed in a cyanobacterial batch culture, thus illuminating the parameters with which cyanobacterial researchers can exploit use to realize their full potential in industrial applications.

microbiology↗

bioRxiv2023

Heterologous production of cyanophycin with Tatumella morbirosei cyanophycin synthetase

Microbial production of biopolymers represents a promising, sustainable alternative to current approaches for plastic production. Cyanophycin synthetase 1 (CphA1) produces cyanophycin - an attractive biopolymer consisting of a poly-L-aspartic acid backbone decorated with L-arginine side groups. In this work, a series of CphA1 enzymes from different bacteria were screened for heterologous cyanophycin production in engineered Escherichia coli, from which it was found that CphA1 from Tatumella morbirosei (TmCphA1) was especially productive. TmCphA1 was capable of supporting up to ~2-fold greater yields of insoluble cyanophycin than any other tested CphA1 enzymes, including 10.8-times more than CphA1 from Synechocystis sp. PCC6308. Finally, using a bench-scale bioreactor, cyanophycin production by TmCphA1-expressing E. coli reached up to 1.9 g per liter of culture by 48 h.

molecular biology↗