Rational Control of Calcium Carbonate Precipitation by Engineered Escherichia coli

Abstract

Ureolytic bacteria (e.g., Sporosarcina pasteurii) can produce calcium carbonate (CaCO 3 ). Tailoring the size and shape of biogenic CaCO 3 may increase the range of useful applications for these crystals. However, wild type Sporosarcina pasteurii is difficult to genetically engineer, limiting control of the organism and its crystal precipitates. Therefore, we designed, constructed, and compared different urease operons and expression levels for CaCO 3 production in engineered Escherichia coli strains. We quantified urease expression and calcium uptake and characterized CaCO 3 crystal phase and morphology for 13 engineered strains. There was a weak relationship between urease expression and crystal size, suggesting that genes surrounding the urease gene cluster affect crystal size. However, when evaluating strains with a wider range of urease expression levels, there was a negative relationship between urease activity and polycrystal size (e.g., larger crystals with lower activity). The resulting range of crystal morphologies created by the rationally designed strains demonstrates the potential for controlling biogenic CaCO 3 precipitation.

Engineered Ureolytic Microorganisms Can Tailor the Morphology and Nanomechanical Properties of Microbial-Precipitated Calcium Carbonate

“…Details of the construction of the engineered E. coli strains and pre-culture growth conditions are described in Liang et al . 24 . In summary, for the E. coli HB101/ure-integration strain, a single copy of the urease gene cluster from S. pasteurii was inserted into the genome using a CRISPR-based technique.…”

“…While S. pasteurii and other native ureolytic microorganisms are difficult to engineer, the genome of E. coli is well-understood and can be readily genetically modified to express urease with different activities. We previously demonstrated that engineered ureolytic E. coli produces CaCO 3, and constructed several strains with a range of urease activities and corresponding precipitation kinetics 24 . Our engineered strain E. coli HB101/ure-integration, with relatively low urease activity and slow precipitation kinetics, produced larger calcite crystals than other E. coli strains with greater urease activity, such as E. coli HB101/pBU11.…”

Engineering living building materials for enhanced bacterial viability and mechanical properties

“…PCC 7002 (referred to as Synechococcus in the following discussion) growing and inducing MICP as a result of the CO 2 -concentrating mechanism (CCM) (Jansson and Northen, 2010). The other two pathways took advantage of urea degradation, which is known to create a local alkaline environment and induce more MICP (e.g., ureolytic Sporosarcina pasteurii, Stocks-Fischer et al, 1999); in this study a bioengineered Escherichia coli strain (referred to as E. coli in the following discussion) that expresses urease operon from S. pasteurii (HB101:pBU11) (Liang et al, 2018;Bachmeier et al, 2002) and the Synechococcus sp. PCC 7002, which is also capable of ureolytic activity (Sakamoto et al, 1998), were grown with additional urea to induce MICP.…”

“…PCC 7002, which is also capable of ureolytic activity (Sakamoto et al, 1998), were grown with additional urea to induce MICP. This initial study investigates the engineered ureolytic strain E. coli HB101:pBU11, which has the advantage to allow multiple genetic designs capable of greater rational control of CaCO 3 precipitation (Liang et al, 2018), leading to variable crystal morphology and nanomechanical properties (Heveran et al, 2019), opening exciting new possibilities for tailoring LBM properties. The three pathways are summarized in Figure 1.…”

Hybrid Living Capsules Autonomously Produced by Engineered Bacteria

“…To demonstrate this ability in our system, we sought to produce capsules capable of biomineralization. To achieve this, we used a commercially available E. coli strain, HB101, transformed with the plasmid pBR322-Ure, which contains the urease gene cluster from the soil bacterium S. pasteurii (49). Urease catalyzes hydrolysis of urea, which leads to the formation of carbonate ions and an (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.…”